Methods and Systems for Molecular Fingerprinting

ABSTRACT

This invention relates in general to a method for molecular fingerprinting. The method can be used for forensic identification (e.g. DNA fingerprinting, especially by VNTR), bacterial typing, and human/animal pathogen diagnosis. More particularly, molecules such as polynucleotides (e.g. DNA) can be assessed or sorted by size in a microfabricated device that analyzes the polynucleotides according to restriction fragment length polymorphism. In a microfabricated device according to the invention, DNA fragments or other molecules can be rapidly and accurately typed using relatively small samples, by measuring for example the signal of an optically-detectable (e.g., fluorescent) reporter associated with the polynucleotide fragments.

This application claims priority under 35 U.S.C. §119(e) to copendingU.S. provisional patent application Ser. No. 60/194,422 filed on Apr. 4,2000. The present application is also a continuation-in-part ofcopending U.S. patent application Ser. Nos. 08/932,774 and 09/325,667,filed on Sep. 23, 1997 and May 21, 1999, respectively. Each of theseprior applications is hereby incorporated by reference in its entirety.

1. FIELD OF THE INVENTION

This invention relates in general to a method for molecularfingerprinting. The method can be used for forensic identification (e.g.DNA fingerprinting, especially by VNTR), bacterial typing, andhuman/animal pathogen diagnosis. More particularly, molecules such aspolynucleotides (e.g. DNA) can be assessed or sorted by size in a microfabricated device that analyzes the polynucleotides according torestriction fragment length polymorphism. In a microfabricated deviceaccording to the invention, DNA fragments or other molecules can berapidly and accurately typed using relatively small samples, bymeasuring for example the signal of an optically-detectable (e.g.,fluorescent) reporter associated with the polynucleotide fragments.

More generally, the invention relates to a method of analyzing orsorting molecules such as polynucleotides (e.g., DNA) by size or someother characteristic. In particular, the invention relates to a methodof analyzing and/or sorting individual polynucleotide molecules in amicrofabricated device by measuring the signal of anoptically-detectable (e.g., fluorescent, ultraviolet, radioactive orcolor change) reporter associated with the molecules. These methods anddevices can also be adapted to analyze or sort cells or particles.

The devices and methods of the invention are advantageous, particularlyin comparison with conventional gel electrophoresis techniques. Forexample, the invention provides less costly and more rapid equipment,can use smaller molecular samples, is less labor-intensive and is morereadily automated. The invention is also advantageously flexible.Additional functions can be incorporated into the design as desired,such as in-line digestion, separation, etc.

2. BACKGROUND OF THE INVENTION

When DNA is broken into fragments using restriction enzymes, each ofwhich cuts the DNA in a known way, the resulting DNA fragments orpolypeptides of different sizes produce a unique pattern or profilewhich can be used to uniquely identify the source of the DNA molecules.In the invention, a reporter or other measurable signal varies as afunction of molecule size, and in this way profiles based on size can beefficiently generated and compared, particularly on a small scale and inan automated or semi-automated fashion.

Methods enabling the matching of unidentified tissue samples to specificindividuals have wide application in many fields. For DNAfingerprinting, commonly used methods include RFLP analysis (53, 54),variable nucleotide tandem repeats (55), and microsatellites (56). Withthe possible exception of monozygotic twins, each individual in thehuman population has a unique genetic composition which can be used tospecifically identify each individual. This phenomenon has allowed lawenforcement officials to use DNA sequence variation to determine, forexample, whether a forensic sample was derived from any givenindividual. The fields of forensic and medical serology, paternitytesting, and tissue and sample origin have seen increasing use of suchtechniques, including the forensic and diagnostic use of DNA sequencevariation, e.g., statistical evaluations based on satellite sequencesand variable number of tandem repeats (VNTRS) or amplified fragmentlength polymorphisms (AMP-FLPS). These methods are being used in crimelaboratories, courts, hospitals and research and testing labs. Inclusionprobabilities stated by the laboratories performing the analyses in suchcases often exceed 1:1,000,000. That is, only one individual in onemillion is predicted, on a statistical basis, to have a given DNA“fingerprint” obtained by analyzing a pattern of DNA fragments generatedaccording to these techniques.

The first implementation of DNA typing in forensics was Jeffreys' use ofa multilocus DNA probe “fingerprint” that identified a suspect in amurder case in England. (55) In the United States, DNA profiling hasbeen established using a battery of unlinked highly polymorphic singlelocus VNTR probes. (57) The use of these batteries of probes permits thedevelopment of a composite DNA profile for an individual. These profilescan be compared to databases, for example using the principles ofHardy-Weinberg to determine the probability of a match between a suspectand an unknown forensic sample.

Although these methods have markedly improved the power of the forensicand medical scientists to distinguish between individuals, they sufferfrom a number of shortcomings including a lack of sensitivity, theabsence of internal controls, expense, time intensity, relatively largesample size, an inability to perform precise allele (gene pair)identification, and problems with identifying degraded DNA samples.

For example, the most frequently used method for forensic identificationis the “Southern” hybridization technique, which has been widely used inforensic identification and medical diagnosis. Also called a “Southernblot,” this technique treats an extracted molecule (a DNA sample) with arestriction endonuclease, an enzyme that cuts a polynucleotide chainwherever a specific and relatively short sequence of nucleic acids inthe chain occurs. Examples of well known restriction enzymes used inthis way are the endocucleases HaeIII, EcoRI, HpaI and HindIII. In DNAfingerprinting, restriction sites are typically used to isolate VNTRs(variable number of tandem repeats), which are regions in which a shortsequence of DNA has been repeated a number of times. The number ofrepeating units within these regions vary between individuals, and whencut with a restriction endonuclease result in multiple fragments ofdifferent size called RFLPs (restriction fragment length polymorphisms).These fragments can be used as a “fingerprint” because they vary innumber and size from one individual to another.

The resulting nucleotide fragments (i.e. the RFLPs) are separated bysize via gel electrophoresis, in which different sized charged moleculesare separated by their different rates of movement through a stationarygel under the influence of an electric current. Followingelectrophoresis, the separated nucleotides are denatured and transferredto the surface of a nylon membrane by blotting; the so-called “Southern.Blot”. The Southern Blot is then incubated in a solution containing aradioactive single locus probe under conditions of temperature and saltconcentration that favor hybridization. (A single locus probe is alsocalled a “primer.”) The locations of radioactive probe hybridization onthe Southern Blot are detected and recorded via X-ray film or some otherdetection technique, thus providing a “profile” of the nucleotide.(Hybridization is used to pull out VNTR fragments, i.e. to separate themfrom irrelevant fragments.) In this approach, sample DNA is digested,and the resulting fragments are separated by size using gelelectrophoresis. The separated fragments are transferred to a membraneby blotting, and are subjected to primer hybridization. (58)

This technique is time-consuming, labor intensive, and the gel may havea limited resolving power, making it potentially difficult to interpretthe results. Another disadvantage is that these techniques generallyrequire the use of a polymerase chain reaction (PCR) to multiply thepolynucleotide in the sample. That is, the conventional tests are notvery sensitive, and require relatively large DNA samples which often arenot available. In such cases the sample concentration is increased to ameaningful detectable level by PCR. While this addresses some problemsof sensitivity and sample degradation, PCR has been open to challengebecause of possible sample contamination, and consequent undesirableamplification of contaminants leading to unreliable results. PCRapproaches are also difficult to multiplex. For example, the probes andprimers must be chosen with care, and generally only one set can beused. The sample may be consumed by one round of PCR, and different setsof probes or primers may require different reaction conditions, such astemperature. A simpler, more powerful technique is needed, which canaccommodate small samples, does not rely on PCR, and which makes use ofthe most recent advances in DNA technology.

As described herein, the invention addresses these problems. Enpreferred embodiments a DNA sample is digested, primers are used toextend specifically desired DNA regions (e.g. VNTRs), without successiverounds of PCR, and highly sensitive or specific reporter molecules, suchas fluorescently-labeled single nucleotides, are used to efficientlydetermine the length of the resulting DNA. A microfabricated ormicrofluidic device may be used to implement these techniques, forexample to separate and optically detect labeled fragments.

The identification and separation of nucleic acid fragments by size,such as in sequencing of DNA or RNA, is a widely used technique in manyfields, including molecular biology, biotechnology, and medicaldiagnostics. The most frequently used method for such separation is gelelectrophoresis, in which different sized charged molecules areseparated by their different rates of movement through a stationary gelunder the influence of an electric current. Gel electrophoresis presentsseveral disadvantages, however. The process can be time consuming, andresolution is typically about 10%. Efficiency and resolution decrease asthe size of fragments increases; molecules larger than 40,000 base pairsare difficult to process, and those larger than 10 million base pairscannot be distinguished.

Methods have been proposed for determination of the size of nucleic acidmolecules based on the level of fluorescence emitted from moleculestreated with a fluorescent dye. See Keller, et al., 1995 (31); Goodwin,et al., 1993 (28); Castro, et. al., 1993 (27); and Quake, et al., 1999(59). Castro (27) describes the detection of individual molecules insamples containing either uniformly sized (48 Kbp) DNA molecules or apredetermined 1:1 ratio of molecules of two different sizes (48 Kbp and24 Kbp). A resolution of approximately 12-15% was achieved between thesetwo sizes. There is no discussion of sorting or isolating thedifferently sized molecules.

In order to provide a small diameter sample stream, Castro (27) uses a“sheath flow” technique wherein a sheath fluid hydrodynamically focusesthe sample stream from 100 μm to 20 μm. This method requires that theradiation exciting the dye molecules, and the emitted fluorescence, musttraverse the sheath fluid, leading to poor light collection efficiencyand resolution problems caused by lack of uniformity. Specifically, thismethod results in a relatively poor signal-to-noise ratio of thecollected fluorescence, leading to inaccuracies in the sizing of the DNAmolecules.

Goodwin (28) mentions the sorting of fluorescently stained DNA moleculesby flow cytometry. This method, however, employs costly and cumbersomeequipment, and requires atomization of the nucleic acid solution intodroplets, with the requirement that each droplet contains at most oneanalyte molecule. Furthermore, the flow velocities required forsuccessful sorting of DNA fragments were determined to be considerablyslower than used in conventional flow cytometry, so the method wouldrequire adaptations to conventional equipment. Sorting a usable amount(e.g., 100 ng) of DNA using such equipment would take weeks; if notmonths, for a single run, and would generate inordinately large volumesof DNA solution requiring additional concentration and/or precipitationsteps.

Quake (59) relates to a single molecule sizing microfabricated device(SMS) for sorting polynucleotides or particles by size, charge or otheridentifying characteristics, for example, characteristics that can beoptically detected. The invention includes a fluorescence activatedsorter (FAS), and methods for analyzing and sorting polynucleotides bymeasuring a signal produced by an optically-detectable (e.g.,fluorescent, ultraviolet or color change) reporter associated with themolecules. These methods and microfabricated devices allow for highsensitivity, no cross-contamination, and lower cost than conventionalgel techniques. In one embodiment of the invention, it has beendiscovered that devices of this kind can be advantageously designed foruse in molecular fingerprinting applications, such as DNAfingerprinting.

It is thus desirable to provide a method of rapidly analyzing andsorting differently sized nucleic acid molecules with high resolution,using simple and inexpensive equipment. In a microfabricated system, ashort optical path length is desirable to reduce distortion and improvesignal-to-noise of detected radiation. Ideally, sorting of fragments canbe carried out using any size-based criteria.

3. SUMMARY OF THE INVENTION

The invention provides a molecular fingerprinting method and system,including for example microfabricated devices for sortingreporter-labeled polynucleotides or polynucleotide molecules by size.

An object of the present invention is a method for DNA fingerprintingusing synthetic repeat polymorphisms.

An additional object of the present invention is a method foridentifying the source of DNA in a forensic or medical sample.

A further object of the present invention is to provide an automated DNAprofiling assay. This case be used, for example for DNA mapping, e.g. ofBAC or YAC libraries.

An additional object of the present invention is to provide a kit fordetecting synthetic repeat polymorphisms.

In accomplishing these and other objectives, the invention provides amethod for molecular fingerprinting using a synthetic version ofrestriction fragment length polymorphism. The method includes choosingat random a short (20-50 bp) sequence of the polynucleotide that is afixed distance away from a restriction site. This can be repeated anynumber of times for enhanced statistical discrimination, with differentlocations in the polynucleotide and different distances to a restrictionsite. Thus, a unique set of fragments can be generated, resulting in afingerprint that can be obtained without relying on naturally occurringrepeat sequences or restriction sites.

The method also provides for identification of a fingerprint in asample. To identify a fingerprinted polynucleotide in a sample, anoligonucleotide (i.e. a short polynucleotide probe) is synthesized tocomplement the randomly chosen sequences. The probes are mixed with thesample along with nucleotide triphosphates and polymerase. Thenucleotides can be fluorescently labeled. Through this technique a setof fluorescent strands of polynucleotide will be synthesized. Eachcomplementary strand is cut with restriction enzymes to yield apolynucleotide of a fixed length. The polynucleotides can then be sized,either by gel electrophoresis or in a single molecule sizing device(SMS). One oligonucleotide probe derived from a references sample can beused, resulting in one complementary strand in a test sample containingmatching sequences. If multiple oligonucleotides are designed, thereaction can be multiplexed and the different length fragments can beresolved into a multiple fragment fingerprint that can be compared tothe standard or reference fingerprint. Preferably, a digestion isperformed before enzyme/primer extension to prevent non-specific bindingof primers. A six-base cutter (digestion enzyme) is particularlypreferred to cut the sample into fragments of tens of thousands of basepairs. Alternatively, digestion after extension to fix the length can beperformed.

A number of variations and modifications to this technique will beapparent to the practitioner of ordinary skill. For example, instead ofusing labeled nucleotides, complementary polynucleotides can bepost-stained with an intercalating dye. Another variation is to useaffinity purification to pull down the fragment of interest, i.e., usingbiotinylated oligonucleotides and streptavidin coated magnetic beads.

In a preferred embodiment, a micro fabricated device is used fordetecting or sorting the nucleotide fragments in a fingerprint based onsize. The SMS device is fast, allowing analysis in as little as 10minutes, and requires only femtograms of material, thus, the SMS deviceprovides relatively high sensitivity without the need for PCR.

Mircofabricated Device. The device includes a chip having a substratewith at least one microfabricated analysis unit. Each analysis unitincludes a main channel, having at one end a sample inlet, having alongits length a detection region, and having, adjacent and downstream ofthe detection region, an outlet or a branch point discrimination regionleading to a plurality of branch channels originating at thediscrimination region and in communication with the main channel. Theanalysis unit also provides a stream of solution, preferably continuous,containing the molecules and passing through the detection region, suchthat on average only one molecule occupies the detection region at anygiven time. The level of reporter from each molecule is measured as itpasses within the detection region. If desired, the molecule is directedto a selected branch channel based on the level of reporter.

In a preferred embodiment, the substrate is planar, and contains amicrofluidic chip made from a silicone elastomer impression of an etchedsilicon wafer according replica methods in soft-lithography (11). In oneembodiment, the channels meet to form a “T” (T junction). A Y-shapedjunction, and other shapes and geometries may also be used. A detectionregion is typically upstream from the branch point. Molecules or cellsare diverted into one or another outlet channel based on a predeterminedcharacteristic that is evaluated as each cell passes through thedetection region. The channels are preferably sealed to contain theflow, for example by fixing a transparent coverslip, such as glass, overthe chip, to cover the channels while permitting optical examination ofone or more channels or regions, particularly the detection region. In apreferred embodiment the coverslip is pyrex, anodically bonded to thechip.

Other devices such as electrophoresis chips may also be used. Exemplarydevices are described in U.S. Pat. Nos. 6,042,709; 5,965,001; 5,948,227;5,880,690; and 6,007,690.

Channel Dimensions. The channels in a molecular analysis device arepreferably between about 1 μm and about 20 μm in width and between about1 μm and about 20 μm in depth, and the detection region has a volume ofbetween about 1 fl and about 1 pl. In a cell analysis device thechannels are preferably between about 1 and 500 microns in width andbetween about 1 and 500 microns in depth, and the detection region has avolume of between about 1 fl and 100 nl. In preferred embodiments, thedevice includes a transparent (e.g., glass) cover slip bonded to thesubstrate and covering the channels to form the roof of the channels.The channels may be of any dimensions suitable to accommodate thelargest dimension of the molecules to be analyzed.

Manifolds. A device which contains a plurality of analysis units mayfurther include a plurality of manifolds, the number of such manifoldstypically being equal to the number of branch channels in one analysisunit, to facilitate collection of molecules from corresponding branchchannels of the different analysis units.

Flow of Molecules. In one embodiment, the molecules are directed orsorted by electroosmotic force. A pair of electrodes apply an electricfield or gradient across the discrimination region that is effective tomove the flow of molecules through the device. In a sorting embodimentthe electrodes can be switched to direct a particular molecule into aselected branch channel based on the amount of reporter signal detectedfrom that molecule. In another embodiment, a flow of molecules ismaintained through the device via a pump or pressure differential, and avalve structure can be used at the branch point effective to permit eachmolecule to enter only one selected branch channel. Alternatively, avalve can be placed in one or more channels downstream of the branchpoint to allow or curtail flow through each channel. In a related,pressure can be adjusted at the outlet of each branch channel effectiveto allow or curtail flow through the channel.

Optical Detection. Preferably the molecules are optically detectablewhen passing through the detection region. For example the molecules maybe labeled with a reporter, for example a fluorescent reporter. Theoptically detectable signal can be measured, and generally isproportional to or is a function of a characteristic of the molecules,such as size or molecular weight. A fluorescent reporter, generating aquantitative optical signal can be used. Fluorescent reporters areknown, and can be associated with molecules such as polynucleotidesusing known techniques.

In a preferred molecular fingerprinting embodiment, the reporter labelis a fluorescently-labeled single nucleotides, such as fluorescein-dNTP,rhodamine-dNTP, Cy3-dNTP, Cy5-dNTP, where dNTP represents dATP, dTTP,dUTP or dCTP. The reporter can also be chemically-modified singlenucleotides, such as biotin-dNTP. Alternatively, chemicals can be usedthat will react with an attached functional group such as biotin.

Sorting Molecules. In another aspect, the invention includes a method ofisolating polynucleotides having a selected size. The method includes:a) flowing a continuous stream of solution containing reporter-labeledpolynucleotides through a channel comprising a detection region having aselected volume, where the concentration of the molecules in thesolution is such that the molecules pass through the detection regionone-by-one, c) determining the size of each molecule as it passesthrough the detection region by measuring the level of the reporter, d)in the continuous stream of solution, diverting (i) molecules having theselected size into a first branch channel, and (ii) molecules not havingthe selected size into a second branch channel. Polynucleotides divertedinto any channel can be collected as desired.

Flow Control. In preferred embodiments, the concentration ofpolynucleotides in the solution is between about 10 fM and about 1 nMand the detection region volume is between about 1 fl and about 1 pl.The molecules can be diverted, for example, by transient application ofan electric field effective to bias (i) a molecule having the selectedsize (e.g., between about 100 bp and about 10 mb) to enter one branchchannel, and (ii) a molecule not having the selected size to enteranother branch channel. Alternatively, molecules can be directed into aselected channel, based on size, by temporarily blocking the flow inother channels, such that the continuous stream of solution carries themolecule having the selected size into the selected channel. Pumps andvalves may also be used to divert flow, and carry molecules into one oranother channels, and mechanical switches may also be used. Thesemethods can also be used in combination, and likewise molecules can bediverted based on whether they have a selected property or size, or donot have that property or size, or exceed or do not exceed a selectedthreshold measurement.

Synchronization. In each embodiment where molecules are measured andthen diverted, as opposed to being measured only, the molecules aredetected and measured one-by-one within the detection region, and arediverted one-by-one into the appropriate channels, by coordinating orsynchronizing the diversion of flow with the detection step and with theflow entering the detection, as described for example in more detailbelow. In certain embodiments the flow rate may be adjusted, for exampledelayed, to maintain efficient detection and switching, and as describedbelow the flow may in some cases be temporarily reversed to improveaccuracy.

Sizing Molecules. In yet another aspect, the invention includes a methodof sizing polynucleotides in solution. This method includes: a) flowinga continuous stream of solution containing reporter-labeledpolynucleotides through a microfabricated channel comprising a detectionregion having a selected volume, where the concentration of themolecules in the solution is such that most molecules pass through thedetection region one by one, and b) determining the size of eachmolecule as it passes through the detection region by measuring thelevel of the reporter.

Multiparameter Embodiments. In addition to analyzing or sortingfluorescent and non-fluorescent nucleotide fragments, the SMS can alsoprovide multiparameter analysis. For example, sizing or sorting can bedone according to a window or threshold value, meaning that molecules(e.g. polynucleotides) are selected based on the presence of a signalabove or below a certain value or threshold. There can also be severalpoints of analysis on the same chip for multiple time coursemeasurements.

Thus, the invention provides for the rapid and accurate determination ofthe “profile” of a polynucleotide in high resolution using minimalamounts of material in these simple and inexpensive microfabricateddevices. The methods and devices of the invention can replace or be usedin combination with conventional get based approaches.

4. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a nucleic acid sorting device in accordance with oneembodiment of the invention.

FIG. 2 shows a partial perspective view of a nucleic acid sortingdevice, showing a sample solution reservoir and sample inlet.

FIG. 3A shows one embodiment of a detection region used in a nucleicacid sorting device, having an integrated photodiode detector.

FIG. 3B shows another embodiment of a detection region, having anintegrated photodiode detector, and providing a larger detection volume(than the embodiment of FIG. 3A).

FIGS. 4A-4B show one embodiment of a valve within a branch channel of anucleic acid sorting device, and steps in fabrication of the valve.

FIG. 5A shows one embodiment of a discrimination region used in anucleic acid sorting device, having electrodes disposed within thechannels for electrophoretic discrimination.

FIG. 5B shows another embodiment of a discrimination region used in anucleic acid sorting device, having electrodes disposed forelectroosmotic discrimination.

FIGS. 5C and 5D show two further embodiments of a discrimination region,having valves disposed for pressure electrophoretic separation, wherethe valves are within the branch point, as shown in FIG. 5C, or withinthe branch channels, as shown in FIG. 5D.

FIG. 6 shows a device with analysis units containing a cascade ofdetection and discrimination regions suitable for successive rounds ofpolynucleotide or cell sorting.

FIGS. 7A-7D show initial steps in photolithographic microfabrication ofa nucleic acid sorting device from a silicon wafer, usingphotolithography and several stages of etching.

FIG. 8 shows a schematic representation of a process for obtaining asilicone elastomer impression of a silicon mold to provide amicrofabricated chip according to the invention.

FIG. 9 shows a schematic representation of an apparatus of theinvention, in which a silicone elastomer chip is mounted on an invertedmicroscope for optical detection of a laser-stimulated reporter.Electrodes are used to direct cells in response to the microscopedetection.

FIG. 10 is a photograph of an apparatus of the invention, showing a chipwith an inlet channel and reservoir, a detection region, a branch point,and two outlet channels with reservoirs.

FIGS. 11A and 11B show a sorting scheme according to the invention, indiagrammatic form.

FIGS. 12A and 12B show a reversible sorting scheme according to theinvention.

FIG. 13 shows the results a comparison between a fingerprint for T7phage and a known T7 sample, using the method and a microfabricateddevice of the invention.

FIG. 14 shows the results a comparison between a fingerprint for T7phage and a known lambda phage sample, using the method and amicrofabricated device of the invention.

FIG. 15 shows a comparison between a T7 phage sample and a lambda phagesample against a T7 fingerprint, using a threshold detection algorithmin a microfabricated device of the invention.

5. DETAILED DESCRIPTION OF THE INVENTION 5.1. Definitions

The terms used in this specification generally have their ordinarymeanings in the art, within the context of the invention, and in thespecific context where each term is used. Certain terms are discussedbelow, or elsewhere in the specification, to provide additional guidanceto the practitioner in describing the devices and methods of theinvention and how to make and use them. For convenience, certain termsare highlighted, for example using italics and/or quotation marks. Theuse of highlighting has no influence on the scope and meaning of a term;the scope and meaning of a term is the same, in the same context,whether or not it is highlighted. It will be appreciated that the samething can be said in more than one way. Consequently, alternativelanguage and synonyms may be used for any one or more of the termsdiscussed herein, nor is any special significance to be placed uponwhether or not a term is elaborated or discussed herein. Synonyms forcertain terms are provided. A recital of one or more synonyms does notexclude the use of other synonyms. The use of examples anywhere in thisspecification, including examples of any terms discussed herein, isillustrative only, and in no way limits the scope and meaning of theinvention or of any exemplified term. Likewise, the invention is notlimited to the preferred embodiments.

General Definitions. As used herein, the term “isolated” means that thereferenced material is removed from the environment in which it isnormally found. Thus, an isolated biological material can be free ofcellular components, i.e., components of the cells in which the materialis found or produced. In the case of nucleic acid molecules, an isolatednucleic acid includes a PCR product, an isolated mRNA, a cDNA, or arestriction fragment. In another embodiment, an isolated nucleic acid ispreferably excised from the chromosome in which it may be found, andmore preferably is no longer joined to non-regulatory, non-codingregions, or to other genes, located upstream or downstream of the genecontained by the isolated nucleic acid molecule when found in thechromosome. In yet another embodiment, the isolated nucleic acid lacksone or more introns. Isolated nucleic acid molecules include sequencesinserted into plasmids, cosmids, artificial chromosomes, and the like.Thus, in a specific embodiment, a recombinant nucleic acid is anisolated nucleic acid. An isolated protein may be associated with otherproteins or nucleic acids, or both, with which it associates in thecell, or with cellular membranes if it is a membrane-associated protein.An isolated organelle, cell, or tissue is removed from the anatomicalsite in which it is found in an organism. An isolated material may be,but need not be, purified.

The term “purified” as used herein refers to material that has beenisolated under conditions that reduce or eliminate the presence ofunrelated materials, i.e., contaminants, including native materials fromwhich the material is obtained. For example, a purified protein ispreferably substantially free of other proteins or nucleic acids withwhich it is associated in a cell; a purified nucleic acid molecule ispreferably substantially free of proteins or other unrelated nucleicacid molecules with which it can be found within a cell. As used herein,the term “substantially free” is used operationally, in the context ofanalytical testing of the material. Preferably, purified materialsubstantially free of contaminants is at least 50% pure; morepreferably, at least 90% pure, and more preferably still at least 99%pure. Purity can be evaluated by chromatography, gel electrophoresis,immunoassay, composition analysis, biological assay, and other methodsknown in the art.

Methods for purification are well-known in the art. For example, nucleicacids can be purified by precipitation, chromatography (includingpreparative solid phase chromatography, oligonucleotide hybridization,and triple helix chromatography), ultracentrifugation, and other means.Polypeptides and proteins can be purified by various methods including,without limitation, preparative disc-gel electrophoresis, isoelectricfocusing, HPLC, reversed-phase HPLC, gel filtration, ion exchange andpartition chromatography, precipitation and salting-out chromatography,extraction, and countercurrent distribution. For some purposes, it ispreferable to produce the polypeptide in a recombinant system in whichthe protein contains an additional sequence tag that facilitatespurification, such as, but not limited to, a poly histidine sequence, ora sequence that specifically binds to an antibody, such as FLAG and GST.The polypeptide can then be purified from a crude lysate of the hostcell by chromatography on an appropriate solid-phase matrix.Alternatively, antibodies produced against the protein or againstpeptides derived therefrom can be used as purification reagents. Cellscan be purified by various techniques, including centrifugation, matrixseparation (e.g., nylon wool separation), panning and otherimmunoselection techniques, depletion (e.g., complement depletion ofcontaminating cells), and cell sorting (e.g., fluorescence activatedcell sorting which is also referred to as FACS). Other purificationmethods are possible. A purified material may contain less than about50%, preferably less than about 75%, and most preferably less than about90%, of the cellular components with which it was originally associated.The “substantially pure” indicates the highest degree of purity whichcan be achieved using conventional purification techniques known in theart.

A “sample” as used herein refers to a biological material which can betested, e.g., for the presence of CK-2 polypeptides or CK-2 nucleicacids, e.g., to identify cells that specifically express the CK-2 geneand its gene product. Such samples can be obtained from any source,including tissue, blood and blood cells, including circulatinghematopoietic stem cells (for possible detection of protein or nucleicacids), plural effusions, cerebrospinal fluid (CSF), ascites fluid, andcell culture. In preferred embodiments samples are obtained from bonemarrow.

Non-human animals include, without limitation, laboratory animals suchas mice, rats, rabbits, hamsters, guinea pigs, etc.; domestic animalssuch as dogs and cats; and, farm animals such as sheep, goats, pigs,horses, and cows.

In preferred embodiments, the terms “about” and “approximately” shallgenerally mean an acceptable degree of error for the quantity measuredgiven the nature or precision of the measurements. Typical, exemplarydegrees of error are within 20 percent (%), preferably within 10%, andmore preferably within 5% of a given value or range of values.Alternatively, and particularly in biological systems, the terms “about”and “approximately” may mean values that are within an order ofmagnitude, preferably within 5-fold and more preferably within 2-fold ofa given value. Numerical quantities given herein are approximate unlessstated otherwise, meaning that the term “about” or “approximately” canbe inferred when not expressly stated.

The term “molecule” means any distinct or distinguishable structuralunit of matter comprising one or more atoms, and includes, for example,polypeptides and polynucleotides.

As used herein, the term “cell” means any cell or cells (e.g.,biological cells) as well as viruses or any other particle having amicroscopic size; e.g., a size that similar to (such as having the sameorder of magnistude as) the Size of a biological cell. The terms celltherefore encompasses both prokaryotic and eukaryotic cells; includingbacteria, fungi, plant and animal cells. Cells are typically spherical,but can also be elongated, flattened, deformable and asymmetrical, i.e.,non-spherical. The size or diameter of a cell typically ranges fromabout 0.1 to 120 microns, and typically is from about 1 to 50 microns. Acell may be living or dead. Since the microfabricated device of theinvention is directed to sorting materials having a size similar to abiological cell (e.g. about 0.1 to 120 microns) any material having asize similar to a biological cell can be characterized and sorted usingthe microfabricated device of the invention: Thus; the term cell shallfurther include microscopic beads (such as chromatogrophic andfluorescent beads), liposomes, emulsions, or any other encapsulatingbiomaterials and porous materials. Non-limiting examples include latex,glass, or paramagnetic beads; and vesicles such as emulsions andliposomes, and other porous materials such as silica beads. Beadsranging in size from 0.1 micron to 1 mm can also be used, for example insorting a library of compounds produced by combinatorial chemistry. Asused herein, a cell may be charged or uncharged. For example, chargedbeads may be used to facilitate flow or detection, or as a reporter.Biological cells, living or dead, may be charged for example by using asurfactant, such as SDS (sodium dodecyl sulfate).

A “reporter” is any molecule, or a portion thereof, that is detectable,or measurable, for example, by optical detection. In addition, thereporter associates with a molecule or cell or with a particular markeror characteristic of the molecule or cell, or is itself detectable, topermit identification of the molecule or cell, or the presence orabsence of a characteristic of the molecule or cell. In the case ofmolecules such as polynucleotides such characteristics include size,molecular weight, the presence or absence of particular constituents ormoeties (such as particular nucleotide sequences or restrictions sites).The term “label” can be used interchangeably with “reporter”. Thereporter is typically a dye, fluorescent, ultraviolet, orchemiluminescent agent, chromophore, or radio-label, any of which may bedetected with or without some kind of stimulatory event, e.g., fluorescewith or without a reagent. Typical reporters for molecularfingerprinting include without limitation fluorescently-labeled singlenucleotides such as fluorescein-dNTP, rhodamine-dNTP, Cy3-dNTP,Cy5-dNTP, where dNTP represents dATP, dTTP, dUTP or dCTP. The reportercan also be chemically-modified single nucleotides, such as biotin-dNTP.Alternatively, chemicals can be used that react with an attachedfunctional group such as biotin.

A “marker” is a characteristic of a molecule or cell that is detectableor is made detectable by a reporter, or which may be coexpressed with areporter. For molecules, a marker can be particular constituents ormoeties, such as restrictions sites or particular nucleic acid sequencesin the case of polynucleotides. The marker may be directly or indirectlyassociated with the reporter or can itself be a reporter. Thus, a markeris generally a distinguishing feature of a molecule, and a reporter isgenerally an agent which directly or indirectly identifies or permitsmeasurement of a marker. These terms may, however, be usedinterchangeably.

The term “flow” means any movement of liquid or solid through a deviceor in a method of the invention, and encompasses without limitation anyfluid stream, and any material moving with, within or against thestream, whether or not the material is carried by the stream. Forexample, the movement of molecules or cells through a device or in amethod of the invention, e.g. through channels of a microfluidic chip ofthe invention, comprises a flow. This is so, according to the invention,whether or not the molecules or cells are carried by a stream of fluidalso comprising a flow, or whether the molecules or cells are caused tomove by some other direct or indirect force or motivation, and whetheror not the nature of any motivating force is known or understood. Theapplication of any force may be used to provide a flow, includingwithout limitation, pressure, capillary action, electro-osmosis,electrophoresis, dielectrophoresis, optical tweezers, and combinationsthereof, without regard for any particular theory or mechanism ofaction, so long as molecules or cells are directed for detection,measurement or sorting according to the invention.

An “inlet region” is an area of a microfabricated chip that receivesmolecules or cells for detection measurement or sorting. The inletregion may contain an inlet channel, a well or reservoir, an opening,and other features which facilitate the entry of molecules or cells intothe device. A chip may contain more than one inlet region if desired.The inlet region is in fluid communication with the main channel and isupstream therefrom.

An “outlet region” is an area of a microfabricated chip that collects ordispenses molecules or cells after detection, measurement or sorting. Anoutlet region is downstream from a discrimination region, and maycontain branch channels or outlet channels. A chip may contain more thanone outlet region if desired.

An “analysis unit” is a microfabricated substrate, e.g., amicrofabricated chip, having at least one inlet region, at least onemain channel, at least one detection region and at least one outletregion. Sorting embodiments of the analysis unit include adiscrimination region and/or a branch point, e.g. downstream of thedetection region, that forms at least two branch channels and two outletregions. A device of the invention may comprise a plurality of analysisunits.

A “main channel” is a channel of the chip of the invention which permitsthe flow of molecules or cells past a detection region for detection(identification), measurement, or sorting. En a chip designed forsorting; the main channel also comprises a discrimination region. Thedetection and discrimination regions can be placed or fabricated intothe main channel. The main channel is typically in fluid communicationwith an inlet channel or inlet region, which permit the flow ofmolecules or cells into the main channel. The main channel is alsotypically in fluid communication with an outlet region and optionallywith branch channels, each of which may have an outlet channel or wastechannel. These channels permit the flow of cells out of the mainchannel.

A “detection region” is a location within the chip, typically within themain channel where molecules or cells to be identified, measured orsorted are examined on the basis of a predetermined characteristic. In apreferred embodiment, molecules or cells are examined one at a time, andthe characteristic is detected or measured optically, for example, bytesting for the presence or amount of a reporter. For example, thedetection region is in communication with one or more microscopes,diodes, light stimulating devices, (e.g., lasers), photomultipliertubes, and processors (e.g., computers and software), and combinationsthereof, which cooperate to detect a signal representative of acharacteristic, marker, or reporter, and to determine and direct themeasurement or the sorting action at the discrimination region. Insorting embodiments the detection region is in fluid communication witha discrimination region and is at, proximate to, or upstream of thediscrimination region.

A “discrimination region” or “branch point” is a junction of a channelwhere the flow of molecules or cells can change direction to enter oneor more other channels, e.g., a branch channel, depending on a signalreceived in connection with an examination in the detection region.Typically, a discrimination region is monitored and/or under the controlof a detection region, and therefore a discrimination region may“correspond” to such detection region. The discrimination region is incommunication with and is influenced by one or more sorting techniquesor flow control systems, e.g., electric, electro-osmotic, (micro-)valve, etc. A flow control system can employ a variety of sortingtechniques to change or direct the flow of molecules or cells into apredetermined branch channel.

A “branch channel” is a channel which is in communication with adiscrimination region and a main channel. Typically, a branch channelreceives molecules or cells depending on the molecule or cellcharacteristic of interest as detected by the detection region andsorted at the discrimination region. A branch channel may be incommunication with other channels to permit additional sorting.Alternatively, a branch channel may also have an outlet region and/orterminate with a well or reservoir to allow collection or disposal ofthe molecules or cells.

The term “forward sorting” or flow describes a one-direction flow ofmolecules or cells, typically from an inlet region (upstream) to anoutlet region (downstream), and preferably without a change indirection, e.g., opposing the “forward” flow. Preferably, molecules orcells travel forward in a linear fashion, i.e., in single file. Apreferred “forward” sorting algorithm consists of running molecules orcells from the input channel to the waste channel, until a molecule orcell is identified to have an optically detectable signal (e.g.fluorescence) that is above a pre-set threshold, at which point voltagesare temporarily changed to electroosmotically divert the molecule or tothe collection channel.

The term “reversible sorting” or flow describes a movement or flow thatcan change, i.e., reverse direction, for example, from a forwarddirection to an opposing backwards direction. Stated another way,reversible sorting permits a change in the direction of flow from adownstream to an upstream direction. This may be useful for moreaccurate sorting, for example, by allowing for confirmation of a sortingdecision, selection of particular branch channel, or to correct animproperly selected channel.

Different algorithms for sorting in the microfluidic device can beimplemented by different programs, for example under the control of apersonal computer. As an example, consider a pressure-switched schemeinstead of electroosmotic flow. Electro-osmotic switching is virtuallyinstantaneous and throughput is limited by the highest voltage that canbe applied to the sorter (which also affects the run time through iondepletion effects). A pressure switched-scheme does not require highvoltages and is more robust for longer runs. However, mechanicalcompliance in the system is likely to cause the fluid switching speed tobecome rate-limiting with the “forward” sorting program. Since the fluidis at low Reynolds number and is completely reversible, when trying toseparate rare molecules or cells one can implement a sorting algorithmthat is not limited by the intrinsic switching speed of the device. Themolecules or cells flow at the highest possible static (non-switching)speed from the input to the waste. When an interesting molecule or cellis detected, the flow is stopped. By the time the flow stops, themolecule or cell may be past the junction and part way down the wastechannel. The system is then run backwards at a slow (switchable) speedfrom waste to input, and the molecule or cell is switched to thecollection channel when it passes through the detection region. At thatpoint, the molecule or cell is “saved” and the device can be run at highspeed in the forward direction again. Similarly, an device of theinvention that is used for analysis, without sorting, can be run inreverse to re-read or verify the detection or analysis made for one ormore molecules or cells in the detection region. This “reversible”analysis or sorting method is not possible with standard gelelectrophoresis technologies (for molecules) nor with conventional FACSmachines (for cells). Reversible algorithms are particularly useful forcollecting rare molecules or cells or making multiple time coursemeasurements of a molecule or single cell.

Molecular Biology Definitions. In accordance with the present invention,there may be employed conventional molecular biology, microbiology andrecombinant DNA techniques within the skill of the art. Such techniquesare explained fully in the literature. See, for example, Sambrook,Fitsch & Maniatis, Molecular Cloning: A Laboratory Manual, SecondEdition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y. (referred to herein as “Sambrook et al., 1989”); DNA Cloning: APractical Approach, Volumes I and II (D. N. Glover ed. 1985);Oligonucleotide Synthesis (M. J. Gait ed. 1984); Nucleic AcidHybridization (B. D. Hames & S. J. Higgins, eds. 1984); Animal CellCulture (R. I. Freshney, ed. 1986); Immobilized Cells and Enzymes (IRLPress, 1986); B. E. Perbal, A Practical Guide to Molecular Cloning(1984); F. M. Ausubel et al. (eds.), Current Protocols in MolecularBiology, John Wiley & Sons, Inc. (1994).

The term “polymer” means any substance or compound that is composed oftwo or more building blocks ('mers') that are repetitively linkedtogether. For example, a “dimer” is a compound in which two buildingblocks have been joined together; a “trimer” is a compound in whichthree building blocks have been joined together; etc.

The term “polynucleotide” or “nucleic acid molecule” as used hereinrefers to a polymeric molecule having a backbone that supports basescapable of hydrogen bonding to typical polynucleotides, wherein thepolymer backbone presents the bases in a manner to permit such hydrogenbonding in a specific fashion between the polymeric molecule and atypical polynucleotide (e.g., single-stranded DNA). Such bases aretypically inosine, adenosine, guanosine, cytosine, uracil and thymidine.Polymeric molecules include “double stranded” and “single stranded” DNAand RNA, as well as backbone modifications thereof (for example,methylphosphonate linkages).

Thus, a “polynucleotide” or “nucleic acid” sequence is a series ofnucleotide bases (also called “nucleotides”), generally in DNA and RNA,and means any chain of two or more nucleotides. A nucleotide sequencefrequently carries genetic information, including the information usedby cellular machinery to make proteins and enzymes. The terms includegenomic DNA, cDNA, RNA, any synthetic and genetically manipulatedpolynucleotide, and both sense and antisense polynucleotides. Thisincludes single- and double-stranded molecules; i.e., DNA-DNA, DNA-RNA,and RNA-RNA hybrids as well as “protein nucleic acids” (PNA) formed byconjugating bases to an amino acid backbone. This also includes nucleicacids containing modified bases, for example, thio-uracil, thio-guanineand fluoro-uracil.

The polynucleotides herein may be flanked by natural regulatorysequences, or may be associated with heterologous sequences, includingpromoters, enhancers, response elements, signal sequences,polyadenylation sequences, introns, 5′- and 3′-non-coding regions andthe like. The nucleic acids may also be modified by many means known inthe art. Non-limiting examples of such modifications includemethylation, “caps”, substitution of one or more of the naturallyoccurring nucleotides with an analog, and internucleotide modificationssuch as, for example, those with uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoroamidates, carbamates, etc.) andwith charged linkages (e.g., phosphorothioates, phosphorodithioates,etc.). Polynucleotides may contain one or more additional covalentlylinked moieties, such as proteins (e.g., nucleases, toxins, antibodies,signal peptides, poly-L-lysine, etc.), intercalators acridine, psoralen,etc.), chelators (e.g., metals, radioactive metals, iron, oxidativemetals, etc.) and alkylators to name a few. The polynucleotides may bederivatized by formation of a methyl or ethyl phosphotriester or analkyl phosphoramidite linkage. Furthermore, the polynucleotides hereinmay also be modified with a label capable of providing a detectablesignal, either directly or indirectly. Exemplary labels includeradioisotopes, fluorescent molecules, biotin and the like. Othernon-limiting examples of modification which may be made are provided,below, in the description of the present invention.

As used herein, the term “oligonucleotide” refers to a nucleic acid,generally of at least 10, preferably at least 15, and more preferably atleast 20 nucleotides, preferably no more than 100 nucleotides, that ishybridizable to a genomic DNA molecule, a cDNA molecule, or an mRNAmolecule encoding a gene, mRNA, cDNA, or other nucleic acid of interest.Oligonucleotides can be labeled, e.g., with ³²P-nucleotides ornucleotides to which a label, such as biotin or a fluorescent dye (forexample, Cy3 or Cy5) has been covalently conjugated. In one embodiment,a labeled oligonucleotide can be used as a probe to detect the presenceof a nucleic acid. In another embodiment, oligonucleotides (one or bothof which may be labeled) can be used as PCR primers, either for cloningfull length or a fragment of a particular nucleic acid (e.g., aparticular gene or a particular gene sequence), or to detect thepresence of particular nucleic acids (e.g., of a particular gene or aparticular gene sequence). Generally, oligonucleotides are preparedsynthetically, preferably on a nucleic acid synthesizer. Accordingly,oligonucleotides can be prepared with non-naturally occurringphosphoester analog bonds, such as thioester bonds, etc.

Specific non-limiting examples of synthetic nucleotides (includingpolynucleotides and oligonucleotides) envisioned for this inventioninclude, in addition to the nucleic acid moieties described above,oligonucleotides that contain phosphorothioates, phosphotriesters,methyl phosphonates, short chain alkyl, or cycloalkyl intersugarlinkages or short chain heteroatomic or heterocyclic intersugarlinkages. Most preferred are those with CH₂—NH—O—CH₂, CH₂—N(CH₃)—O—CH₂,CH₂—O—N(CH₃)—CH₂, CH₂—N(CH₃)—N(CH₃)—CH₂ and O—N(CH₃)—CH₂—CH₂ backbones(where phosphodiester is O—PO₂—O—CH₂). U.S. Pat. No. 5,677,437 describesheteroaromatic olignucleoside linkages. Nitrogen linkers or groupscontaining nitrogen can also be used to prepare oligonucleotide mimics(U.S. Pat. Nos. 5,792,844 and 5,783,682). U.S. Pat. No. 5,637,684describes phosphoramidate and phosphorothioamidate oligomeric compounds.Also envisioned are oligonucleotides having morpholino backbonestructures (U.S. Pat. No. 5,034,506). In other embodiments, such as thepeptide-nucleic acid (PNA) backbone, the phosphodiester backbone of theoligonucleotide may be replaced with a polyamide backbone, the basesbeing bound directly or indirectly to the aza nitrogen atoms of thepolyamide backbone (Nielsen et al., Science 254:1497, 1991). Othersynthetic oligonucleotides may contain substituted sugar moietiescomprising one of the following at the 2′ position: OH, SH, SCH₃, F,OCN, O(CH₂)_(n)NH₂ or O(CH₂)_(n)CH₃ where n is from 1 to about 10; C₁ toC₁₀ lower alkyl, substituted lower alkyl, alkaryl or aralkyl; Cl; Br;CN; CF₃; OCF₃; O—; S—, or N-alkyl; O—, S—, or N-alkenyl; SOCH₃; SO₂CH₃;ONO₂; NO₂; N₃; NH₂; heterocycloalkyl; heterocycloalkaryl;aminoalkylamino; polyalkylamino; substitued silyl; a fluorescein moiety;an RNA cleaving group; a reporter group; an intercalator; a group forimproving the pharmacokinetic properties of an oligonucleotide; or agroup for improving the pharmacodynamic properties of anoligonucleotide, and other substituents having similar properties.Oligonucleotides may also have sugar mimetics such as cyclobutyls orother carbocyclics in place of the pentofuranosyl group. Nucleotideunits having nucleosides other than adenosine, cytidine, guanosine,thymidine and uridine, such as inosine, may be used in anoligonucleotide molecule.

A “polypeptide” is a chain of chemical building blocks called aminoacids that are linked together by chemical bonds called “peptide bonds”.The term “protein” refers to polypeptides that contain the amino acidresidues encoded by a gene or by a nucleic acid molecule (e.g., an mRNAor a cDNA) transcribed from that gene either directly or indirectly.Optionally, a protein may lack certain amino acid residues that areencoded by a gene or by an mRNA. For example, a gene or mRNA moleculemay encode a sequence of amino acid residues on the N-terminus of aprotein (i.e., a signal sequence) that is cleaved from; and thereforemay not be part of, the final protein. A protein or polypeptide,including an enzyme, may be a “native” or “wild-type”, meaning that itoccurs in nature; or it may be a “mutant”, “variant” or “modified”,meaning that it has been made, altered, derived, or is in some waydifferent or changed from a native protein or from another mutant.

“Amplification” of a polynucleotide, as used herein, denotes the use ofpolymerase chain reaction (PCR) to increase the concentration of aparticular DNA sequence within a mixture of DNA sequences. For adescription of PCR see Saiki et al., Science 1988, 239:487.

“Chemical sequencing” of DNA denotes methods such as that of Maxam andGilbert (Maxam-Gilbert sequencing; see Maxam & Gilbert, Proc. Natl.Acad. Sci. U.S.A. 1977, 74:560), in which DNA is cleaved usingindividual base-specific reactions.

“Enzymatic sequencing” of DNA denotes methods such as that of Sanger(Sanger et al., Proc. Natl. Acad. Sci. U.S.A. 1977, 74:5463) andvariations thereof well known in the art, in a single-stranded DNA iscopied and randomly terminated using DNA polymerase.

A “gene” is a sequence of nucleotides which code for a functional “geneproduct”. Generally, a gene product is a functional protein. However, agene product can also be another type of molecule in a cell, such as anRNA (e.g., a tRNA or a rRNA). For the purposes of the present invention,a gene product also refers to an mRNA sequence which may be found in acell. For example, measuring gene expression levels according to theinvention may correspond to measuring mRNA levels. A gene may alsocomprise regulatory (i.e., non-coding) sequences as well as codingsequences. Exemplary regulatory sequences include promoter sequences,which determine, for example, the conditions under which the gene isexpressed. The transcribed region of the gene may also includeuntranslated regions including introns, a 5′-untranslated region(5′-UTR) and a 3′-untranslated region (3′-UTR).

A “coding sequence” or a sequence “encoding” an expression product, suchas a RNA, polypeptide, protein or enzyme, is a nucleotide sequence that,when expressed, results in the production of that RNA, polypeptide,protein or enzyme; i.e., the nucleotide sequence “encodes” that RNA orit encodes the amino acid sequence for that polypeptide, protein orenzyme.

A “promoter sequence” is a DNA regulatory region capable of binding RNApolymerase in a cell and initiating transcription of a downstream (3′direction) coding sequence. For purposes of defining the presentinvention, the promoter sequence is bounded at its 3′ terminus by thetranscription initiation site and extends upstream (5′ direction) toinclude the minimum number of bases or elements necessary to initiatetranscription at levels detectable above background. Within the promotersequence will be found a transcription initiation site (convenientlyfound, for example, by mapping with nuclease S1), as well as proteinbinding domains (consensus sequences) responsible for the binding of RNApolymerase.

A coding sequence is “under the control of” or is “operativelyassociated with” transcriptional and translational control sequences ina cell when RNA polymerase transcribes the coding sequence into RNA,which is then trans-RNA spliced (if it contains introns) and, if thesequence encodes a protein, is translated into that protein.

The term “express” and “expression” means allowing or causing theinformation in a gene or DNA sequence to become manifest, for exampleproducing RNA (such as rRNA or mRNA) or a protein by activating thecellular functions involved in transcription and translation of acorresponding gene or DNA sequence. A DNA sequence is expressed by acell to form an “expression product” such as an RNA (e.g., a mRNA or arRNA) or a protein. The expression product itself, e.g., the resultingRNA or protein, may also said to be “expressed” by the cell.

The term “transfection” means the introduction of a foreign nucleic acidinto a cell. The term “transformation” means the introduction of a“foreign” (i.e., extrinsic or extracellular) gene, DNA or RNA sequenceinto a host cell so that the host cell will express the introduced geneor sequence to produce a desired substance, in this invention typicallyan RNA coded by the introduced gene or sequence, but also a protein oran enzyme coded by the introduced gene or sequence. The introduced geneor sequence may also be called a “cloned” or “foreign” gene or sequence,may include regulatory or control sequences (e.g., start, stop,promoter, signal, secretion or other sequences used by a cell's geneticmachinery). The gene or sequence may include nonfunctional sequences orsequences with no known function. A host cell that receives andexpresses introduced DNA or RNA has been “transformed” and is a“transformant” or a “clone”. The DNA or RNA introduced to a host cellcan come from any source, including cells of the same genus or speciesas the host cell or cells of a different genus or species.

The terms “vector”, “cloning vector” and “expression vector” mean thevehicle by which a DNA or RNA sequence (e.g., a foreign gene) can beintroduced into a host cell so as to transform the host and promoteexpression (e.g., transcription and translation) of the introducedsequence. Vectors may include plasmids, phages, viruses, etc. and arediscussed in greater detail below.

A “cassette” refers to a DNA coding sequence or segment of DNA thatcodes for an expression product that can be inserted into a vector atdefined restriction sites. The cassette restriction sites are designedto ensure insertion of the cassette in the proper reading frame.Generally, foreign DNA is inserted at one or more restriction sites ofthe vector DNA, and then is carried by the vector into a host cell alongwith the transmissible vector DNA. A segment or sequence of DNA havinginserted or added DNA, such as an expression vector, can also be calleda “DNA construct.” A common type of vector is a “plasmid”, whichgenerally is a self-contained molecule of double-stranded DNA, usuallyof bacterial origin, that can readily accept additional (foreign) DNAand which can readily introduced into a suitable host cell. A largenumber of vectors, including plasmid and fungal vectors, have beendescribed for replication and/or expression in a variety of eukaryoticand prokaryotic hosts. The term “host cell” means any cell of anyorganism that is selected, modified, transformed, grown or used ormanipulated in any way for the production of a substance by the cell.For example, a host cell may be one that is manipulated to express aparticular gene, a DNA or RNA sequence, a protein or an enzyme. Hostcells can further be used for screening or other assays that aredescribed infra. Host cells may be cultured in vitro or one or morecells in a non-human animal (e.g., a transgenic animal or a transientlytransfected animal).

The term “expression system” means a host cell and compatible vectorunder suitable conditions, e.g. for the expression of a protein codedfor by foreign DNA carried by the vector and introduced to the hostcell. Common expression systems include E. coli host cells and plasmidvectors, insect host cells such as Sf9, Hi5 or S2 cells and Baculovirusvectors, Drosophila cells (Schneider cells) and expression systems, fishcells and expression systems (including, for example, RTH-149 cells fromrainbow trout, which are available from the American Type CultureCollection and have been assigned the accession no. CRL-1710) andmammalian host cells and vectors.

The term “heterologous” refers to a combination of elements notnaturally occurring. For example, the present invention includeschimeric RNA molecules that comprise an rRNA sequence and a heterologousRNA sequence which is not part of the rRNA sequence. In this context,the heterologous RNA sequence refers to an RNA sequence that is notnaturally located within the ribosomal RNA sequence. Alternatively, theheterologous RNA sequence may be naturally located within the ribosomalRNA sequence, but is found at a location in the rRNA sequence where itdoes not naturally occur. As another example, heterologous DNA refers toDNA that is not naturally located in the cell, or in a chromosomal siteof the cell. Preferably, heterologous DNA includes a gene foreign to thecell. A heterologous expression regulatory element is a regulatoryelement operatively associated with a different gene that the one it isoperatively associated with in nature.

The terms “mutant” and “mutation” mean any detectable change in geneticmaterial, e.g., DNA, or any process, mechanism or result of such achange. This includes gene mutations, in which the structure (e.g., DNAsequence) of a gene is altered, any gene or DNA arising from anymutation process, and any expression product (e.g., RNA, protein orenzyme) expressed by a modified gene or DNA sequence. The term “variant”may also be used to indicate a modified or altered gene, DNA sequence,RNA, enzyme, cell, etc.; i.e., any kind of mutant. For example, thepresent invention relates to altered or “chimeric” RNA molecules thatcomprise an rRNA sequence that is altered by inserting a heterologousRNA sequence that is not naturally part of that sequence or is notnaturally located at the position of that rRNA sequence. Such chimericRNA sequences, as well as DNA and genes that encode them, are alsoreferred to herein as “mutant” sequences.

The term “homologous”, in all its grammatical forms and spellingvariations, refers to the relationship between two proteins that possessa “common evolutionary origin”, including proteins from superfamilies(e.g., the immunoglobulin superfamily) in the same species of organism,as well as homologous proteins from different species of organism (forexample, myosin light chain polypeptide, etc.; see, Reeck et al., Cell1987, 50:667). Such proteins (and their encoding nucleic acids) havesequence homology, as reflected by their sequence similarity, whether interms of percent identity or by the presence of specific residues ormotifs and conserved positions.

The term “sequence similarity”, in all its grammatical forms, refers tothe degree of identity or correspondence between nucleic acid or aminoacid sequences that may or may not share a common evolutionary origin(see, Reeck et al., supra). However, in common usage and in the instantapplication, the term “homologous”, when modified with an adverb such as“highly”, may refer to sequence similarity and may or may not relate toa common evolutionary origin.

In specific embodiments, two nucleic acid sequences are “substantiallyhomologous” or “substantially similar” when at least about 80%, and morepreferably at least about 90% or at least about 95% of the nucleotidesmatch over a defined length of the nucleic acid sequences, as determinedby a sequence comparison algorithm known such as BLAST, FASTA, DNAStrider, CLUSTAL, etc. An example of such a sequence is an allelic orspecies variant of the specific genes of the present invention.Sequences that are substantially homologous may also be identified byhybridization, e.g., in a Southern hybridization experiment under, e.g.,stringent conditions as defined for that particular system.

Similarly, in particular embodiments of the invention, two amino acidsequences are “substantially homologous” or “substantially similar” whengreater than 80% of the amino acid residues are identical, or whengreater than about 90% of the amino acid residues are similar (i.e., arefunctionally identical). Preferably the similar or homologouspolypeptide sequences are identified by alignment using, for example,the GCG (Genetics Computer Group, Program Manual for the GCG Package,Version 7, Madison Wis.) pileup program; or using any of the programsand algorithms described above (e.g., BLAST, FASTA, CLUSTAL, etc.).

A nucleic acid molecule is “hybridizable” to another nucleic acidmolecule, such as a cDNA, genomic DNA, or RNA, when a single strandedform of the nucleic acid molecule can anneal to the other nucleic acidmolecule under the appropriate conditions of temperature and solutionionic strength (see Sambrook et al., supra). The conditions oftemperature and ionic strength determine the “stringency” of thehybridization. For preliminary screening for homologous nucleic acids,low stringency hybridization conditions, corresponding to a T_(m)(melting temperature) of 55° C., can be used, e.g., 5×SSC, 0.1% SDS,0.25% milk, and no formamide; or 30% formamide, 5×SSC, 0.5% SDS).Moderate stringency hybridization conditions correspond to a higherT_(m), e.g., 40% formamide, with 5× or 6×SCC. High stringencyhybridization conditions correspond to the highest T_(m), e.g., 50%formamide, 5× or 6×SCC. SCC is a 0.15M NaCl, 0.015M Na-citrate.Hybridization requires that the two nucleic acids contain complementarysequences, although depending on the stringency of the hybridization,mismatches between bases are possible. The appropriate stringency forhybridizing nucleic acids depends on the length of the nucleic acids andthe degree of complementation, variables well known in the art. Thegreater the degree of similarity or homology between two nucleotidesequences, the greater the value of T_(m) for hybrids of nucleic acidshaving those sequences. The relative stability (corresponding to higherT_(m)) of nucleic acid hybridizations decreases in the following order:RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100 nucleotidesin length, equations for calculating T_(m) have been derived (seeSambrook et al., supra, 9.50-9.51). For hybridization with shorternucleic acids, i.e., oligonucleotides, the position of mismatchesbecomes more important, and the length of the oligonucleotide determinesits specificity (see Sambrook et al., supra, 11.7-11.8). A minimumlength for a hybridizable nucleic acid is at least about 10 nucleotides;preferably at least about 15 nucleotides; and more preferably the lengthis at least about 20 nucleotides.

In a specific embodiment; the term “standard hybridization conditions”refers to a T_(m) of 55° C., and utilizes conditions as set forth above.In a preferred embodiment, the T_(m) is 60° C.; in amore preferredembodiment, the T_(m) is 65° C. En a specific embodiment, “highstringency” refers to hybridization and/or washing conditions at 68° C.in 0.2×SSC, at 42° C. in 50% formamide, 4×SSC, or under conditions thatafford levels of hybridization equivalent to those observed under eitherof these two conditions.

Suitable hybridization conditions for oligonucleotides (e.g., foroligonucleotide probes or primers) are typically somewhat different thanfor full-length nucleic acids (e.g., full-length cDNA), because of theoligonucleotides' lower melting temperature. Because the meltingtemperature of oligonucleotides will depend on the length of theoligonucleotide sequences involved, suitable hybridization temperatureswill vary depending upon the oligonucleotide molecules used. Exemplarytemperatures may be 37° C. (for 14-base oligonucleotides), 48° C. (for17-base oligoncucleotides), 55° C. (for 20-base oligonucleotides) and60° C. (for 23-base oligonucleotides). Exemplary suitable hybridizationconditions for oligonucleotides include washing in 6×SSC/0.05% sodiumpyrophosphate, or other conditions that afford equivalent levels ofhybridization.

5.2. Overview of the Invention

The invention provides a method and system for molecular fingerprinting,and devices for molecular fingerprinting, including microfabricatedmicrofluidic devices for evaluating or sorting molecules according tosize. More particularly, polynucleotides such as DNA samples can befragmented, for example using endonucleases, to produce a set offragments that vary in size. The size distribution of these fragments(e.g. the number of fragments of each size over a range of sizes) mayuniquely identify the source of the sample. Some or all of the fragmentscan be selected to serve as a “fingerprint” of the sample. Further,fragments comprising the fingerprint can be labeled, for example with areporter molecule such as fluorescent marker, so that the they can bemore readily detected, measured or sorted.

These measurements can be detected by any suitable means, preferablyoptical, and can be ‘stored for example in’ a computer as arepresentation of the fragments comprising the fingerprint Depending onthe strategy for producing the fragments which comprise a fingerprint,oligonucleotide probes of known composition and length may be used to“tag” or label the fragments. For example, probes having sequences thatare complementary to each of the fragments can be made by combining thefragments with labeled nucleotide bases in the presence of a polymerase,which is an enzyme that assembles a single strand of complementarypolynucleotide using another strand (i.e. a fingerprint fragment) as atemplate. The nucleotide bases used to make these probes may beradioactive, or can be labeled with a fluorescent marker, or with someother readily detectable reporter. The resulting probes can be used torecord a fingerprint of the sample, by detecting and measuring the leverof reporter as an indication of size, or by sorting the probes accordingto size.

Labeled or unlabeled probes can also be used to “fish out” matchingpolynucleotides from a test sample containing unknown DNA orpolynucleotides. Under appropriate hybridizing conditions, probes willbind to matching fragments in a sample. This can provide a way to testfor a match, for example when the probes comprising a fingerprinthybridize to complementary fragments in the sample.

According to one aspect of the invention polynucleotides can befingerprinted using a synthetic version of restriction fragment lengthpolymorphism. The method includes choosing at random a short (20-50 bp)sequence of the polynucleotide that is a fixed distance away from arestriction site. This can be repeated any number of times for enhancedstatistical discrimination, with different locations in thepolynucleotide and different distances to a restriction site. Samplesare digested with a specific restriction enzyme, such as Bg II, EcoR I,Hind III or Xho I. A final mixture of DNA fragments of thousands of basepairs is preferred. En this way a unique fingerprint can be synthesizedwithout relying on naturally occurring repeat sequences or restrictionsites.

The method also provides for identification of the fingerprintednucleotide in a sample. To identify the fingerprinted polynucleotide ina sample, an oligonucleotide probe is synthesized to complement eachrandomly chosen sequence. Each probe is mixed with the test sample alongwith nucleotide triphosphates and polymerase. The nucleotides can befluorescently labeled. Through this technique a fluorescent strand ofpolynucleotide (complementary to each probe) will be synthesized. Eachstrand is then cut with restriction enzymes to yield a polynucleotide ofa fixed length. The polynucleotide can then be sized, either by gelelectrophoresis or in a single molecule sizing device (SMS) (59). Ifmultiple oligonucleotides are designed, the reaction can be multiplexedand the different length fragments can then be resolved into afingerprint that can be compared to a standard fingerprint.

In one aspect of the invention, polynucleotides, e.g., DNA, can bedetected, sized or sorted dynamically in a continuous flow stream ofmicroscopic dimensions based for example on molecular weight, using amicrofabricated polynucleotide sorting device. The polynucleotides,suspended in a suitable carrier fluid (e.g., ddH₂0 or TE), areintroduced into an inlet end of a narrow channel in the sorting device.The molecular weight of each molecule is calculated from the intensityof signal from an optically-detectable reporter incorporated into orassociated with the polynucleotide molecule as the molecule passesthrough a “detection window” or “detection region” in the device.

In a sorter embodiment, molecules having a molecular weight fallingwithin a selected range are diverted into a selected output or “branch”channel of the device. The sorted polynucleotide molecules may becollected from the output channels and used in subsequent manipulations.

According to another aspect of the invention, a device such as describedabove, but not necessarily including components for sorting themolecules, can be used to measure or quantify the size range ofpolynucleotides in a sample, and store or feed this information into aprocessor or computer for subsequent analysis or display, e.g., as asize distribution histogram. Such a device enables the generation of thetype of polynucleotide fragment length data now commonly obtained fromanalytical gels, such as agarose or polyacrylamide gels, or fromSouthern blot results, in a fraction of the time required forpreparation and analysis of gels, and using a substantially smalleramount of sample.

5.3. Microfluidic Chip Architecture and Methods

A molecular or cell analyzer or sorter according to the inventioncomprises at least one analysis unit having an inlet region incommunication with a main channel, a detection region within orcoincident with a portion of the main channel, and a detector associatedwith the detection region. Sorter embodiments also have a discriminationregion or branch point in communication with the main channel and withbranch channels, and a flow control responsive to the detector. Thebranch channels may each lead to an outlet region and to a well orreservoir. The inlet region may also communicate with a well orreservoir. As each molecule or cell passes into the detection region, itis examined for a predetermined characteristic (i.e. using thedetector), and a corresponding signal is produced, for exampleindicating that “yes” the characteristic is present, or “no” it is not.The signal may correspond to a characteristic qualitatively orquantitatively. That is, the amount of the signal can be measured andcan correspond to the degree to which a characteristic is present. Forexample, the strength of the signal may indicate the size of themolecule, or the potency or amount of an enzyme expressed by a cell. Inresponse to the signal, data can be collected and/or a flow control canbe activated to divert a molecule or cell into one branch channel oranother. Thus, molecules or cells within the discrimination region canbe sorted into an appropriate branch channel according to a signalproduced by the corresponding examination at the detection region.Optical detection of molecule or cell characteristics is preferred, forexample directly or by use of a reporter associated with acharacteristic chosen for sorting. However, other detection techniquesmay also be employed.

A variety of channels for sample flow and mixing can be microfabricatedon a single chip and can be positioned at any location on the chip asthe detection and discrimination or sorting points, e.g., for kineticstudies (12, 14). A plurality of analysis units of the invention may becombined in one device. Microfabrication applied according to theinvention eliminates the dead time occurring in conventional gelelectrophoresis or flow cytometric kinetic studies, and achieves abetter time-resolution. Furthermore, linear arrays of channels on asingle chip, i.e., a multiplex system, can simultaneously detect andsort a sample by using an array of photomultiplier tubes (PMT) forparallel analysis of different channels (15). This arrangement can beused to improve throughput or for successive sample enrichment, and canbe adapted to provide a very high throughput to the microfluidic devicesthat exceeds the capacity permitted by conventional flow sorters.Moreover, microfabrication permits other technologies to be integratedor combined on a single chip, such as PCR (21), moving molecules orcells using optical tweezer/trapping (16-18), transformation of cells byelectroporation (19), μTAS (22), and DNA hybridization (6). Detectorsand/or light filters that are used to detect molecule or cellcharacteristics or their reporters can also be fabricated directly onthe chip.

A device of the invention can be microfabricated with a sample solutionreservoir or well at the inlet region, which is typically in fluidcommunication with an inlet channel. A reservoir may facilitateintroduction of molecules or cells into the device and into the sampleinlet channel of each analysis unit. An inlet region may have anopening, such as in the floor of the microfabricated chip, to permitentry of the sample into the device. The inlet region may also contain aconnector adapted to receive a suitable piece of tubing, such as liquidchromatography or HPLC tubing, through which a sample may be supplied.Such an arrangement facilitates introducing the sample solution underpositive pressure in order to achieve a desired flow rate through thechannels. Outlet channels and wells can be similarly provided.

Substrate and Flow Channels. A typical analysis unit of the inventioncomprises an inlet region that is part of and feeds or communicates witha main channel, which in turn communicates with an outlet (for analysisonly) or with two (or more) branch channels at a junction or branchpoint, forming for example a T-shape or a Y-shape for sorting. Othershapes and channel geometries may be used as desired. The region at orsurrounding the junction can also be referred to as a discriminationregion, however, precise boundaries for the discrimination region arenot required. A detection region is identified within or coincident witha portion of the main channel downstream of the inlet region, and at orupstream of the discrimination region or branch point. Preciseboundaries for the detection region are not required, but are preferred.The discrimination region may be located immediately downstream of thedetection region, or it may be separated by a suitable distanceconsistent with the size of the molecules, the channel dimensions, andthe detection system. It will be appreciated that the channels can haveany suitable shape or cross-section, such as tubular or grooved, and canbe arranged in any suitable manner, so tong as a flow can be directedfrom one channel into at least one of two or more branch channels.

The channels of the invention are microfabricated, for example byetching a silicon chip using conventional photolithography techniques,or using a micromachining technology called “soft lithography”,developed in the late 1990's (11). These and other micro fabricationmethods may be used to provide inexpensive miniaturized devices, and inthe case of soft lithography, can provide robust devices havingbeneficial properties such as improved flexibility, stability, andmechanical strength. When optical detection is employed, the inventionalso provides minimal light scatter from molecule or cell suspension andchamber material. Devices according to the invention are relativelyinexpensive and easy to set up. They can also be disposable, whichgreatly relieves many of the concerns of gel electrophoresis (formolecules) and for sterilization and permanent adsorption of particlesunto the flow chambers and channels of conventional FACS machines (forcells). Using these kinds of techniques, microfabricated fluidic devicescan replace the conventional gel electrophoresis and fluidic flowchambers of the prior art.

A microfabricated device of the invention is preferably fabricated froma silicon microchip or silicon elastomer. The dimensions of the chip arethose of typical microchips, ranging between about 0.5 cm to about 5 cmper side and about 1 micron to about 1 cm in thickness. The devicecontains at least one analysis unit containing a main channel havingdetection and discrimination regions. Preferably a device also containsat least one inlet region (which may contain an inlet channel) and twoor more outlet regions (which have fluid communication with a branchchannel in each region). It shall be appreciated that the “regions” and“channels” are in fluid communication wish each other, and thereforethey may overlap, i.e., there may be no clear boundary where a region orchannel begins or ends. A microfabricated device can be covered with amaterial having transparent properties, e.g., a glass coverslip topermit detection of a reporter for example by an optical device, such asan optical microscope.

The dimensions of the channels and in particular of the detection regionare influenced by the size of the molecules or cells under study. Forpolynucleotides, which are large by molecular standards, a typicallength or diameter is about 3.4 angstroms per base pair. Thus, a DNA 49kpbs long, such as Lambda phage DNA, is about 17 microns long when fullyextended. A typical range of sizes for polynucleotides of the inventionis from about 1 to about 200 kpbs, or about 0.3 to about 70 microns.Detection regions used for detecting molecules have a cross-sectionalarea large enough to allow a desired molecule to pass through withoutbeing substantially slowed down relative to the flow of the solutioncarrying it. To avoid “bottlenecks” and/or turbulence, and promotesingle-file flow, the channel dimensions, particularly in the detectionregion, should generally be at least about twice, preferably at leastabout five times as large per side or in diameter as the diameter of thelargest molecule that will be passing through it.

For molecules such as DNA, the channels in a device are typicallybetween about 1 to about 100 microns (μm) in diameter, with preferredchannel dimensions ranging from about 2 to about 5 microns in width andbetween about 2 and about 4 or 5 microns in depth. Similarly, the volumeof the detection region in a molecular analysis or sorting device may befrom about 1 femtoliter (fl) to about 1 nanoliter (nl). Typically, thedetection region will have a volume between about 10 to about 5000 fl,preferably about 40 or 50 fl to about 1000 or 2000 fl, most preferablyon the order of about 200 fl.

To prevent material from adhering to the sides of the channels, thechannels (and coverslip, if used) may have a coating which minimizesadhesion. Such a coating may be intrinsic to the material from which thedevice is manufactured, or it may be applied after the structuralaspects of the channels have been microfabricated. “TEFLON” is anexample of a coating that has suitable surface properties.

A silicon substrate containing the microfabricated flow channels andother components is preferably covered and sealed, most preferably witha transparent cover, e.g., thin glass or quartz, although other clear oropaque cover materials may be used. When external radiation sources ordetectors are employed, the detection region is covered with a clearcover material to allow optical access to the molecules or cells. Forexample, anodic bonding to a “PYREX” cover slip can be accomplished bywashing both components in an aqueous H₂SO₄/H₂O₂ bath, rinsing in water,and then, for example, heating to about 350 degrees C. while applying avoltage of 450V.

Switching and Flow Control. Electro-osmotic and pressure-driven flow areexamples of methods or systems for flow control, that is, manipulatingthe flow of molecules cells, particles or reagents in one or moredirections and/or into one or more channels of a microfluidic device ofthe invention (8, 12, 13, 23). Other methods may also be used, forexample, electrophoresis and dielectrophoresis. En certain embodimentsof the invention, the flow moves in one “forward” direction, e.g. fromthe inlet region through the main and branch channels to an outletregion. In other embodiments the direction of flow is reversible.Application of these techniques according to the invention provides morerapid and accurate devices and methods for sorting, for example, becausethe sorting occurs at or in a discrimination region that can be placedat or immediately after a detection region. This provides a shorterdistance for molecules or cells to travel, they can move more rapidlyand with less turbulence, and can more readily be moved, examined, andsorted in single file, i.e., one at a time. In a reversible embodiment,potential sorting errors can be avoided, for example by reversing andslowing the flow to re-read or resort a molecule or cell (or a pluralitythereof) before irretrievably committing the molecule or cell to theoutlet or to a particular branch channel.

Without being bound by any theory, electro-osmosis is believed toproduce motion in a stream containing ions, e.g. a liquid such as abuffer, by application of a voltage differential or charge gradientbetween two or more electrodes. Neutral (uncharged) molecules or cellscane be carried by the stream. Electro-osmosis is particularly suitablefor rapidly changing the course, direction or speed of flow.Electrophoresis is believed to produce movement of charged objects in afluid toward one or more electrodes of opposite charge, and away fromone on or more electrodes of like charge. Because of its charged nature(2 charges for each base pair) DNA can be conveniently moved byelectrophoresis in a buffer of appropriate pH.

Dielectrophoresis is believed to produce movement of dielectric objects,which have no net charge, but have regions that are positively ornegatively charged in relation to each other. Alternating,non-homogeneous electric fields in the presence of particles, such asmolecules, cells or beads, cause them to become electrically polarizedand thus to experience dielectrophoretic forces. Depending on thedielectric polarizability of the particles and the suspending medium,dielectric particles will move either toward the regions of high fieldstrength or low field strength. For example, the polarizability ofliving cells depends on their composition, morphology, and phenotype andis highly dependent on the frequency of the applied electrical field.Thus, cells of different types and in different physiological statesgenerally possess distinctly different dielectric properties, which mayprovide a basis for cell separation, e.g., by differentialdielectrophoretic forces. According to formulas provided in Fiedler etal. (13), individual manipulation of single particles requires fielddifferences (inhomogeneties) with dimensions close to the particles.

Manipulation is also dependent on permittivity (a dielectric property)of the particles with the suspending medium. Thus, polymer particles andliving cells show negative dielectrophoresis at high-field frequenciesin water. For example, dielectrophoretic forces experienced by a latexsphere in a 0.5 MV/m field (10V for a 20 micron electrode gap) in waterare predicted to be about 0.2 piconewtons (pN) for a 3.4 micron latexsphere to 15 pN for a 15 micron latex sphere (13). These values aremostly greater than the hydrodynamic forces experienced by the sphere ina stream (about 0.3 pN for a 3.4 micron sphere and 1.5 pN for a 15micron sphere). Therefore, manipulation of individual cells or particlescan be accomplished in a streaming fluid, such as in a cell sorterdevice, using dielectrophoresis. Using conventional semiconductortechnologies, electrodes can be microfabricated onto a substrate tocontrol the force fields in a microfabricated sorting device of theinvention. Dielectrophoresis is particluarly suitable for moving objectsthat are electrical conductors. The use of AC current is preferred, toprevent permanent alignment of ions. Megahertz frequencies are suitableto provide a net alignment, attractive force, and motion over relativelylong distances. E.g. Benecke (49).

Optical tweezers can also be used in the invention to trap and moveobjects, e.g. molecules or cells, with focused beams of light such aslasers. Flow can also be obtained and controlled by providing a pressuredifferential or gradient between one or more channels of a device or ina method of the invention.

Molecules or cells can be moved by direct mechanical switching, e.g.with on-off valves, or by squeezing the channels. Pressure control mayalso be used, for example by raising or lowering an output well tochange the pressure inside the channels on the chip. See e.g. thedevices and methods described in pending. U.S. application Ser. No.08/932,774 filed Sep. 25, 1997; No. 60/108,894 filed Nov. 17, 1998; No.60/086,394 filed May 22, 1998; and No. 09/325,667 filed May 21, 1999(molecular analysis systems). These methods and devices can further beused in combination with the methods and devices described in pendingU.S. application Ser. Nos. 60/141,503 (filed Jun. 28, 1999), 609/147,199(filed Aug. 3, 1999) and 60/186,856 (filed Mar. 3, 2000). Each of thesereferences is hereby incorporated by reference in its entirety.

Detection and Discrimination for Sorting. The detector can be any deviceor method for interrogating a molecule or cell as it passes through thedetection region. Typically, molecules or cells are to be analyzed orsorted according to a predetermined characteristic that is directly orindirectly detectable, and the detector is selected or adapted to detectthat characteristic. A preferred detector is an optical detector, suchas a microscope, which may be coupled with a computer and/or other imageprocessing or enhancement devices to process images or informationproduced by the microscope using known techniques. For example,molecules can be sorted by size or molecular weight. Cells can be sortedfor whether they contain or produce a particular protein, by using anoptical detector to examine each cell for an optical indication of thepresence or amount of that protein. The protein may itself bedetectable, for example by a characteristic fluorescence, or it may belabeled or associated with a reporter that produces a detectable signalwhen the desired protein is present, or is present in at least athreshold amount. There is no limit to the kind or number of molecule orcell characteristics that can be identified or measured using thetechniques of the invention, which include without limitation surfacecharacteristics of the cell and intracellular characteristics, providedonly that the characteristic or characteristics of interest for sortingcan be sufficiently identified and detected or measured to distinguishcells having the desired characteristic(s) from those which do not. Forexample, any label or reporter as described herein can be used as thebasis for sorting molecules or cells, i.e. detecting them to becollected.

In preferred embodiments, the molecules or cells are analyzed and/orseparated based on the intensity of a signal from anoptically-detectable reporter bound to or associated with them as theypass through a detection window or “detection region” in the device.Molecules or cells having an amount or level of the reporter at aselected threshold or within a selected range are diverted into apredetermined outlet or branch channel of the device. The reportersignal is collected by a microscope and measured by a photomultipliertube (PMT). A computer digitizes the PMT signal and controls the flowvia valve action or electro-osmotic potentials. Alternatively, thesignal can be recorded or quantified, as a measure of the reporterand/or its corresponding characteristic or marker, e.g. for purposes ofevaluation without necessarily proceeding to sort the molecules orcells.

In one embodiment, the =chip is mounted on an inverted opticalmicroscope. Fluorescence produced by a reporter is excited using a laserbeam focused on molecules (e.g. DNA) or cells passing through adetection region. Fluorescent reporters include, e.g., rhodamine,fluorescein, Texas red, Cy 3, Cy 5, phycobiliprotein, green fluorescentprotein (GFP), YOY-1, and picogreen. In molecular fingerprintingapplications, the reporter labels are preferably a fluorescently-labeledsingle nucleotides, such as fluorescein-dNTP, rhodamine-dNTP, Cy3-dNTP,Cy5-dNTP, where dNTP represents dATP, dTTP, dUTP or dCTP. The reportercan also be chemically-modified single nucleotides, such as biotin-dNTP.Thus, in one aspect of the invention, the device can determine the sizeor molecular weight of molecules such as polynucleotide fragmentspassing through the detection region, or the presence or degree of someother characteristic indicated by a reporter. If desired, the moleculescan be sorted based on this analysis.

To detect a reporter or determine whether a molecule has a desiredcharacteristic, the detection region may include an apparatus forstimulating a reporter for that characteristic to emit measurable lightenergy, e.g., a light source such as a laser, laser diode,high-intensity lamp, (e.g., mercury lamp), and the like. In embodimentswhere a lamp is used, the channels are preferably shielded from light inall regions except the detection region. In embodiments where a laser isused, the laser can be set to scan across a set of detection regionsfrom different analysis units. In addition, laser diodes may bemicrofabricated into the same chip that contains the analysis units.Alternatively, laser diodes may be incorporated into a second chip(i.e., a laser diode chip) that is placed adjacent to themicrofabricated sorter chip such that the laser light from the diodesshines on the detection region(s).

In preferred embodiments, an integrated semiconductor laser and/or anintegrated photodiode detector are included on the silicon wafer in thevicinity of the detection region. This design provides the advantages ofcompactness and a shorter optical path for exciting and/or emittedradiation, thus minimizing distortion.

Sorting Schemes. According to the invention, molecules or cells aresorted dynamically in a flow stream of microscopic dimensions, based onthe detection or measurement of a characteristic, marker or reporterthat is associated with the molecules or cells. The stream is typicallybut not necessarily continuous, and may be stopped and started,reversed, or changed in speed. Prior to sorting, a liquid that does notcontain sample molecules or cells can be introduced at an inlet regionof the chip (e.g., from an inlet well or channel) and is directedthrough the device by capillary action, to hydrate and prepare thedevice for sorting. If desired, the pressure can be adjusted orequalized for example by adding buffer to an outlet region. The liquidtypically is an aqueous buffer solution, such as ultrapure water (e.g.,18 mega ohm resistivity, obtained for example by column chromatography),ultrapure water, 10 mM Tris HCL and 1 mM EDTA (TE), phosphate buffersaline (PBS), and acetate buffer. Any liquid or buffer that isphysiologically compatible with the population of molecules or cells tobe sorted can be used.

A sample solution containing a mixture or population of molecules orcells in a suitable carrier fluid (such as a liquid or buffer describedabove) is supplied to the inlet region. The capillary force causes thesample to enter the device. The force and direction of flow can becontrolled by any desired method for controlling flow, for example, by apressure differential, by valve action, or by electro-osmotic flow,e.g., produced by electrodes at inlet and outlet channels. This permitsthe movement of the molecules or cells into one or more desired branchchannels or outlet regions.

A “forward” sorting algorithm, according to the invention, includesembodiments where molecules or cells from an inlet channel flow throughthe device to a predetermined branch or outlet channel (which can becalled a “waste channel”), until the level of measurable reporter isabove a pre-set threshold. At that time, the flow is diverted to deliverthe molecule or cell to another channel. For example, in anelectroosmotic embodiment, where switching is virtually instantaneousand throughput is limited by the highest voltage, the voltages aretemporarily changed to divert the chosen molecule or cell to anotherpredetermined outlet channel (which can be called a “collectionchannel”). Sorting, including synchronizing detection of a reporter anddiversion of the flow, can be controlled by various methods includingcomputer or microprocessor control. Different algorithms for sorting inthe microfluidic device can be implemented by different computerprograms, such as programs used in conventional FACS devices for sortingcells. For example, a programmable card can be used to controlswitching, such as a Lab PC 1200 Card, available from NationalInstruments, Austin, Tex. Algorithms as sorting procedures can beprogrammed using C++, LABVIEW, or any suitable software. The method isadvantageous, for example, because conventional gel electrophoresismethods are generally not automated or under computer control.

A “reversible” sorting algorithm can be used in place of a “forward”mode, for example in embodiments where switching speed may be limited.For example, a pressure-switched scheme can be used instead ofelectro-osmotic flow and does not require high voltages and may be morerobust for longer runs. However, mechanical constraints may cause thefluid switching speed to become rate-limiting. In a pressure-switchedscheme the flow is stopped when a molecule or cell of interest isdetected. By the time the flow stops, the molecule or cell may be pastthe branch point and be part-way down the waste channel. In thissituation, a reversible embodiment can be used. The system can be runbackwards at a slower (switchable) speed (e.g., from waste to inlet),and the molecule or cell is then switched to a different channel. Atthat point, a potentially mis-sorted molecule or cell is “saved”, andthe device can again be run at high speed in the forward direction. This“reversible” sorting method is not possible with standard FACS machinesor in gel electrophoresis technologies. FACS machines mostly sortaerosol droplets which cannot be reversed back to the chamber, in orderto be redirected. The aerosol droplet sorter are virtually irreversible.In gel electrophoresis, molecules such as polynucleotides are drawnthrough a gel by an electric current and migrate at different ratesproportional to their molecular weights. Individual molecules can not bereversed through the gel, and indeed, altering the rate or direction ofmigration would prevent meaningful use of the technique. Reversiblesorting is particularly useful for identifying rare molecules or cells(e.g., in molecular evolution and cancer cytological identification), ormolecules or cells that are few in number, which may be misdirected dueto a margin of error inherent to any fluidic device. The reversiblenature of the device of the invention permits a reduction in thispossible error.

A “reversible” sorting method permits multiple time course measurementsof a single molecule or cell. This allows for observations ormeasurements of the same molecule or cell at different times, becausethe flow reverses the molecule or cell back into the detection windowbefore directing it to a downstream channel. Measurements can becompared or confirmed, and changes in molecule or cell properties overtime can be examined, for example in kinetic studies.

When trying to separate molecules or cells in a sample at a very lowratio to the total number of molecules or cells, a sorting algorithm canbe implemented that is not limited by the intrinsic switching speed ofthe device. Consequently, the molecules or cells flow at the highestpossible static (non-switching) speed from the inlet channel to thewaste channel. Unwanted molecules or cells can be directed into thewaste channel at the highest speed possible, and when a desired moleculeor cell is detected, the flow can be slowed down and then reversed, todirect it back into the detection region, from where it can beredirected (i.e. to accomplish efficient switching). Hence the moleculesor cells can flow at the highest possible static speed.

Preferably, the fluid carrying the molecules or cells has a relativelylow Reynolds Number, for example 10⁻². The Reynolds Number represents aninverse relationship between the density and velocity of a fluid and itsviscosity in a channel of given length. More viscous, less dense, slowermoving fluids over a shorter distance will have a lower Reynolds Number,and are easier to divert, stop, start, or reverse without turbulence.Because of the small sizes and slow velocities, microfabricated fluidsystems are often in a low Reynolds number regime (<<1). In this regime,inertial effects, which cause turbulence and secondary flows, arenegligible; viscous effects dominate the dynamics. These conditions areadvantageous for sorting, and are provided by microfabricated devices ofthe invention. Accordingly the microfabricated devices of the inventionare preferably if not exclusively operated at a low or very lowReynold's number. Exemplary sorting schemes are shown diagrammaticallyin FIGS. 11A and B and FIGS. 12A and B.

6. EXAMPLES

The present invention is also described by means of particular examples.However, the use of such examples anywhere in the specification isillustrative only and in no way limits the scope and meaning of theinvention or of any exemplified term. Likewise, the invention is notlimited to any particular preferred embodiments described herein.Indeed, many modifications and variations of the invention will beapparent to those skilled in the art upon reading this specification andcan be made without departing from its spirit and scope. The inventionis therefore to be limited only by the terms of the appended claimsalong with the full scope of equivalents to which the claims areentitled.

6.1. A Microfabricated Polynucleotide Sorting Device

FIG. 1 shows an embodiment of a microfabricated polynucleotide sortingdevice 20 in accordance with the invention. The device is preferablyfabricated from a silicon microchip 22. The dimensions of the chip arethose of typical microchips, ranging between about 0.5 cm to about 5 cmper side and about 0.1 mm to about 1 cm in thickness. The devicecontains a solution inlet 24, two or more solution outlets, such asoutlets 26 and 28, and at least one analysis unit, such as the unit at30.

Each analysis unit includes a main channel 32 having at one end a sampleinlet 34, and downstream of the sample inlet, a detection region 36, anddownstream of the detection region 36 a discrimination region 38. Aplurality of branch channels, such as channels 40 and 42, are in fluidcommunication with and branch out from the discrimination region. Thedimensions of the main and branch channels are typically between about 1μm and 10 μm per side, but may vary at various points to facilitateanalysis, sorting and/or collection of molecules.

In embodiments such as shown in FIG. 1, where the device contains aplurality of analysis units, the device may further contain collectionmanifolds, such as manifolds 44 and 46, to facilitate collection ofsample from corresponding branch channels of different analysis unitsfor routing to the appropriate solution outlet. The manifolds arepreferably microfabricated into different levels of the device, asindicated by the dotted line representing manifold 46. Similarly, suchembodiments may include a sample solution reservoir, such as reservoir48, to facilitate introduction of sample into the sample inlet of eachanalysis unit.

Also included with the device is a processor, such as processor 50. Theprocessor can be integrated into the same chip as contains the analysisunit(s), or it can be separate, e.g., an independent microchip connectedto the analysis unit-containing chip via electronic leads, such as leads52 (connected to the detection region(s) and 54 (connected to thediscrimination region(s)).

As mentioned above, the device may be microfabricated with a samplesolution reservoir to facilitate introduction of a polynucleotidesolution into the device and into the sample inlet of each analysisunit. With reference to FIG. 2, the reservoir is micro fabricated intothe silicon substrate of the chip 62, and is covered, along with thechannels (such as main channel 64) of the analysis units, with a glasscoverslip 66. The device solution inlet comprises an opening 68 in thefloor of the microchip. The inlet may further contain a connector 70adapted to receive a suitable piece of tubing, such as liquidchromatography or HPLC tubing, through which the sample may be supplied.Such an arrangement facilitates introducing the sample solution underpositive pressure, to achieve a desired flow rate through the channelsas described below.

Downstream of the sample inlet of the main channel of each analysis unitis the detection region, designed to detect the level of anoptically-detectable reporter associated with polynucleotides present inthe region. Exemplary embodiments of detection regions in devices of theinvention are shown in FIGS. 3A and 3B.

6.2. Photodiode Detectors

With reference to FIG. 3A, each detection region is formed of a portionof the main channel of an analysis unit and a photodiode, such asphotodiode 72, located in the floor of the main channel. In thisembodiment, the area detectable by the detection region is the circularportion each channel defined by the receptive field of the photodiode inthat channel. The volume of the detection region is the volume of acylinder with a diameter equal to the receptive field of the photodiodeand a height equal to the depth of the channel above the photodiode.

The signals from the photodiodes are carried via output lines 76 to theprocessor (not shown), which processes the signals into valuescorresponding to the length of the polynucleotide giving rise to thesignal. The processor then uses this information, for example, tocontrol active elements in the discrimination region. The processor mayprocess the signals into values for comparison with a predetermined orreference set of values for analysis or sorting.

When more than one detection region is used, the photodiodes in thelaser diode chip are preferably spaced apart relative to the spacing ofthe detection regions in the analysis unit. That is, for more accuratedetection, the photodiodes are placed apart at the same spacing as thespacing of the detection region.

The processor can be integrated into the same chip that contains theanalysis unit(s), or it can be separate, e.g., an independent microchipconnected to the analysis unit-containing chip via electronic leads thatconnect to the detection region(s) and/or to the discriminationregion(s), such as by a photodiode. The processor can be a computer ormicroprocessor, and is typically connected to a data storage unit, suchas computer memory, hard disk, or the like, and/or a data output unit,such as a display monitor, printer and/or plotter.

The types and numbers of molecules or cells, based on detection of areporter associated with or bound to the molecules or cells passingthrough the detection region, can be calculated or determined, and thedata obtained can be stored in the data storage unit. This informationcan then be further processed or routed to the data outlet unit forpresentation, e.g. histograms, of the types of molecules or cells (orlevels of a cell protein, saccharide), or some other characteristic. Thedata can also be presented in real time as the sample is flowing throughthe device.

With reference to FIG. 3B, the photodiode 78 can be larger in diameterthan the width of the main channel, forming a detection region 80 thatis longer (along the length of the main channel 82) than it is wide. Thevolume of such a detection region is approximately equal to thecross-sectional area of the channel above the diode multiplied by thediameter of the diode.

In a preferred sorting embodiment the detection region is connected bythe main channel to the discrimination region. The discrimination regionmay be located immediately downstream of the detection region, or may beseparated by a suitable length of channel. Constraints on the length ofchannel between the detection and discrimination regions are discussedbelow, with respect to the operation of the device. This length istypically between about 1 μm and about 2 cm. The discrimination regionis at the junction of the main channel and the branch channels. Itcomprises the physical location where molecules are directed into aselected branch channel. The means by which the molecules or cells aredirected into a selected branch channel may (i) be present in thediscrimination region, as in, e.g., electrophoretic or microvalve-baseddiscrimination, or (ii) be present at a distant location, as in, e.g.,electroosmotic or flow stoppage-based discrimination.

If desired, the device may contain a plurality of analysis units, i.e.,more than one detection and discrimination region, and a plurality ofbranch channels which are in fluid communication with and branch outfrom the discrimination regions. It will be appreciated that theposition and fate of molecules or cells in the discrimination region canbe monitored by additional detection regions installed, for example,immediately upstream of the discrimination region and/or within thebranch channels immediately downstream of the branch point. Theinformation obtained by the additional detection regions can be used bya processor to continuously revise estimates of the velocity of themolecules or cells in the channels and to confirm that molecules orcells having a selected characteristic enter the desired branch channel.

A group of manifolds (a region consisting of several channels which leadto or from a common channel) can be included to facilitate movement ofsample from the different analysis units, through the plurality ofbranch channels and to the appropriate solution outlet. Manifolds arepreferably microfabricated into the chip at different levels of depth.Thus, devices of the invention having a plurality of analysis units cancollect the solution from associated branch channels of each unit into amanifold, which routes the flow of solution to an outlet. The outlet canbe adapted for receiving, for example, a segment of tubing or a sampletube, such as a standard 1.5 ml centrifuge tube. Collection can also bedone using micropipettes.

6.3. Valve Structures

In an embodiment where pressurized flow is used, valves can be used toblock or unblock the pressurized flow of molecules or cells throughselected channels. A thin cantilever, for example, may be includedwithin a branch point, as shown in FIGS. 4A and 4B, such that it may bedisplaced towards one or the other wall of the main channel, typicallyby electrostatic attraction, thus closing off a selected branch channel.Electrodes are on the walls of the channel adjacent to the end of thecantilever. Suitable electrical contacts for applying a potential to thecantilever are also provided in a similar manner as the electrodes.Because the cantilever in FIG. 4B is parallel to the direction ofetching, it may be formed of a thin layer of silicon by incorporatingthe element into the original photoresist pattern. The cantilever ispreferably coated with a dielectric material such as silicon nitride, asdescribed in Wise, et al., 1995 (35), for example, to prevent shortcircuiting between the conductive surfaces.

Alternatively, a valve may be situated within each branch channel,rather than at the branch point, to close off and terminate pressurizedflow through selected channels. Because the valves are locateddownstream of the discrimination region, the channels in this region maybe formed having a greater width than in the discrimination region,which simplifies the formation of valves.

A valve within a channel may be microfabricated, if desired, in the formof an electrostatically operated cantilever or diaphragm. Techniques forforming such elements are well known in the art (e.g., 24, 29, 35, 36,37). Typical processes, include the use of selectively etchedsacrificial layers in a multilayer structure or, for example, theundercutting of a layer of silicon dioxide via anisotropic etching. Forexample, to form a cantilever within a channel, as illustrated in FIGS.4A and 4B, a sacrificial layer 168 may be formed adjacent to a smallsection of a non-etchable material 170, using known photolithographymethods, on the floor of a channel, as shown in FIG. 4A. Both layers canthen be coated with, for example, silicon dioxide or anothernon-etchable layer, as shown at 172. Etching of the sacrificial layerdeposits the cantilever member 174 within the channel, as shown in FIG.4B. Suitable materials for the sacrificial layer, non-etchable layersand etchant include undoped silicon, p-doped silicon and silicondioxide, and the etchant EDP (ethylene diamine/pyrocatechol),respectively. Because the cantilever in FIG. 4B is parallel to thedirection of etching, it may be formed of a thin layer of silicon byincorporating the element into the original photoresist pattern. Thecantilever is preferably coated with a dielectric material such assilicon nitride, as described in (35) for example, to prevent shortcircuiting between the conductive surfaces.

The width of the cantilever or diaphragm should approximately equal thatof the channel, allowing for movement within the channel. If desired,the element may be coated with a more malleable material, such as ametal, to allow for a better seat. Such coating may also be employed torender a non-conductive material, such as silicon dioxide, conductive.

As above, suitable electrical contacts are provided for displacing thecantilever or diaphragm towards the opposing surface of the channel.When the upper surface is a glass cover plate, as described below,electrodes and contacts may be deposited onto the glass.

It will be apparent to one of skill in the field that other types ofvalves or switches can be designed and fabricated, using well knownphotolithographic or other microfabrication techniques, for controllingflow within the channels of the device. Multiple layers of channels canalso be prepared.

Operation of the valves or charging of the electrodes, in response tothe level of fluorescence measured from an analyte molecule, iscontrolled by the processor, which receives this information from thedetector. All of these components are operably connected in theapparatus, and electrical contacts are included as necessary, usingstandard microchip circuitry.

In preferred embodiments, an integrated semiconductor laser and/or anintegrated photodiode detector are included on the silicon wafer in thevicinity of the detection region. This design provides the advantages ofcompactness and a shorter optical path for exciting and/or emittedradiation, thus minimizing distortion.

The silicon substrate containing the microfabricated flow channels andother components is covered and sealed, preferably with a thin glass orquartz cover, although other clear or opaque cover materials may beused. when external radiation sources or detectors are employed, theinterrogation region is covered with a clear cover material to allowoptical access to the analyte molecules. Anodic bonding to a “PYREX”cover slip may be accomplished by washing both'components in an aqueousH₂SO₄/H₂O₂ bath, rinsing in water, and then heating to about 350° C.while applying a voltage of, e.g., 450V.

6.4. Exemplary Microchip Architectures for Sorting

As illustrated with respect to FIGS. 5A-5D, there are a number of waysin which cells can be routed or sorted into a selected branch channel.

FIG. 5A shows a discrimination region 102, which is suitable forelectrophoretic discrimination as the sorting technique. Thediscrimination region is preceded by a main channel 104. A junctiondivides the main channel into two branch channels 106 and 108. Thediscrimination region 102 includes electrodes 110 and 112, positioned onouter side walls of the branch channels 106 and 108, and which connectto leads 114 and 116. The leads are connected to a voltage source (notshown) incorporated into or controlled by a processor (not shown), asdescribed, infra. The distance (D) between the electrodes is preferablyless than the average distance separating the cells during flow throughthe main channel. The dimensions of the electrodes are typically thesame as the dimensions of the channels in which they are positioned, e.esuch that the electrodes are as high and wide as the channel.

The discrimination region shown in FIG. 5B is suitable for use in adevice that employs electro-osmotic flow, to move the molecules or cellsand bulk solution through the device. FIG. 4B shows a discriminationregion 122 which is preceded by a main channel 124. The main channelcontains a junction that divides the main channel into two branchchannels 126 and 128. An electrode 130 is placed downstream of thejunction of the main channel, for example near the sample inlet of mainchannel. Electrodes are also placed in each branch channel (electrodes132 and 134). The electrode 130 can be negative and electrodes 132 and134 can be positive (or vice versa) to establish bulk solution flowaccording to well-established principles of electro-osmotic flow (25).See, also, U.S. patent application Ser. No. 09/325,667 filed May 21,1999.

After a molecule or cell passes the detection region (not shown) andenters the discrimination region 122 (e.g. between the main channel andthe two branch channels) the voltage to one of the electrodes 132 or 134can be shut off, leaving a single attractive force that acts on thesolution and the molecule or cell to influence it into the selectedbranch channel. As above, the appropriate electrodes are activated afterthe molecule or cell has committed to the selected branch channel inorder to continue bulk flow through both channels. In one embodiment,the electrodes are charged to divert the flow into one branch channel,for example channel 126, which can be called a waste channel. Inresponse to a signal indicating that a molecule or cell has beenidentified or selected for collection, the charge on the electrodes canbe changed to divert the selected molecule or cell into the otherchannel (channel 128), which can be called a collection channel.

In another embodiment of the invention, shown in FIG. 5C, the moleculesor cells are directed into a predetermined branch channel via a valve140 in the discrimination region. The valve 140 comprises a thinextension of material to which a charge can be applied via an electrodelead 142. The valve 140 is shown with both channels open, and can bedeflected to close either branch channel by application of a voltageacross electrodes 144 and 146. A molecule or cell is detected and chosenfor sorting in the detection region (not shown), and can be directed tothe appropriate channel by closing off the other channel, e.g. byapplying, removing or changing a voltage applied to the electrodes. Thevalve can also be configured to close one channel in the presence of avoltage, and to close the other channel in the absence of a voltage.

FIG. 5D shows another embodiment of a discrimination region of theinvention, which uses flow stoppage in one or more branch channels asthe discrimination means. The sample solution moves through the deviceby application of positive pressure at an end where the solution inletis located. Discrimination or routing of the molecules or cells isaffected by simply blocking a branch channel (145 or 148) or a branchchannel sample outlet using valves in a pressure-driven flow (147 or149). Due to the small size scale of the channels and theincompressibility of liquids, blocking the solution flow creates aneffective “plug” in the non-selected branch channel, thereby temporarilyrouting the molecule or cell together with the bulk solution flow intothe selected channel. Valve structures can be incorporated downstreamfrom the discrimination region, which are controlled by the detectionregion, as described herein.

Alternatively, the discrimination function represented in FIG. 5D may becontrolled by changing the hydrostatic pressure at the sample outlets ofone or both branch channels 145 or 148. If the branch channels in aparticular analysis unit have the same resistance to fluid flow, and thepressure at the sample inlet of the main channel of an analysis unit isP, then the fluid flow out of any selected branch channel can be stoppedby applying a pressure P/n at the sample outlet of the desired branchchannel, where n is the number of branch channels in the analysis unit.Accordingly, in an analysis unit having two branch channels, thepressure-applied at the outlet of the branch to be blocked is P/2.

As shown in FIG. 5D, a valve is situated within each branch channel,rather than at the branch point, to close off and terminate pressurizedflow through selected channels. Because the valves are located at apoint downstream from the discrimination region, the channels in thisregion may be formed having a greater width than in the discriminationregion in order to simplify the formation of valves. The width of thecantilever or diaphragm should approximately equal the width of thechannel, allowing for movement within the channel. If desired, theelement may be coated with a more malleable material, such as a metal,to allow for a better seal. Such coating may also be employed to rendera non-conductive material, such as silicon dioxide, conductive. Asabove, suitable electrical contacts are provided for displacing thecantilever or diaphragm towards the opposing surface of the channel.When the upper surface is a glass cover plate, electrodes and contactsmay be deposited onto the glass.

6.5. A Cascade Device

FIG. 6 shows a device with analysis units containing a cascade ofdetection and discrimination regions suitable for successive rounds ofpolynucleotide or cell sorting. Such a configuration may be used, forexample, with a polynucleotide or cellsorting device to generate aseries of samples containing “fractions” of polynucleotides, where eachfraction contains a specific size range of polynucleotide fragments(e.g., the first fraction contains 100-500 bp fragments, the next500-1000 bp fragments, and so on). In a cell sorting device, such acascade configuration may be used to sequentially assay the cell for,e.g., three different fluorescent dyes corresponding to expression ofthree different molecular markers. Samples collected at the outlets ofthe different branch channels contain pools of cells expressing definedlevels of each of the three markers. The number of reporters employed,and therefore the number of markers of interest, can be varied asdesired, e.g. to meet the needs of a particular experiment orapplication.

6.6. Microfabricated Polynucleotide Analysis Device

Also included in the present invention is a microfabricatedpolynucleotide analysis device suitable for quantitation and analysis ofthe size distribution of polynucleotide fragments in solution. Such adevice is a simplified version of the sorting device described above, inthat analysis units in the device need not contain a discriminationregion or branch channels, and the device need not contain a means fordirecting molecules to selected branch channels. Each analysis unitcomprises a single main channel containing a detection region asdescribed above. Since the optics which collect the optical signal(e.g., fluorescence) can be situated immediately adjacent the flowstream (e.g., diode embedded in the channel of a microscope objectiveadjacent a glass coverslip covering the channel), the signal-to-noiseratio of the signal collected using a microfabricated polynucleotideanalysis device of the invention is high relative to other types ofdevices. Specifically, the invention allows, e.g., the use ofoil-immersion high numerical apperature (N.A.) microscope objectives tocollect the light (e.g., 1.4 N.A.). Since the collection of light isproportional to the square of the N.A., a 1.4 N.A. objective providesabout a four-fold better signal than an 0.8 N.A. objective.

6.7. Microfabricated Cell Sorting Device

The invention also includes a microfabricated device for sortingreporter-labeled cells by the level of reporter they contain. The deviceis similar to polynucleotide-sorting devices described above, but isadapted for handling particles on the size scale of cells rather thanmolecules. This difference is manifested mainly in the dimensions of themicrofabricated channels, detection and discrimination regions.Specifically, the channels in the device are typically between about 20μm and about 500 μm in width and between about 20 μm and about 500 μm indepth, to allow for an orderly flow of cells in the channels. Similarly,the volume of the detection region in a cell sorting device is largerthan that of the polynucleotide sorting device, typically being in therange of between about 10 pl and 100 nl. To prevent the cells fromadhering to the sides of the channels, the channels (and coverslip)preferably contain a coating which minimizes cell adhesion. Such acoating may be intrinsic to the material from which the device ismanufactured, or it may be applied after the structural aspects of thechannels have been microfabricated. An exemplary coating has the surfaceproperties of a material such as “TEFLON”.

The device may be used to sort any procaryotic (e.g., bacterial) oreukaryotic (e.g., mammalian) cells which can be labeled (e.g., viaantibodies) with optically-detectable reporter molecules (e.g.,fluorescent dyes). Exemplary mammalian cells include human blood cells,such as human peripheral blood mononuclear cells (PBMCs). The cells canbe labeled with antibodies directed against any of a variety of cellmarker antigens (e.g., HLA DR, CD3, CD4, CD8, CD11a, CD11c, CD 14, CD16,CD20, CD45, CD45RA, CD62L, etc.), and the antibodies can in turn bedetected using an optically-detectable reporter (either via directlyconjugated reporters or via labeled secondary antibodies) according tomethods known in the art.

It will be appreciated that the cell sorting device and method describedabove can be used simultaneously with multiple optically-detectablereporters having distinct optical properties. For example, thefluorescent dyes fluorescein (FITC), phycoerythrin (PE), and “CYCHROME”(Cy5-PE) can be used simultaneously due to their different excitationand emission spectra. The different dyes may be assayed, for example, atsuccessive detection and discrimination regions. Such regions may becascaded as shown in FIG. 6 to provide samples of cells having aselected amount of signal from each dye.

6.8. Microfabrication of a Silicon Device

Analytical devices having microscale flow channels, valves and otherelements can be designed and fabricated from a solid substrate material.Silicon is a preferred substrate material because of the well developedtechnology permitting its precise and efficient fabrication, but othermaterials may be used, including polymers such aspolytetrafluoroethylenes. Micromachining methods well known in the artinclude film deposition processes, such as spin coating and chemicalvapor deposition, laser fabrication or photolithographic techniques, oretching methods, which may be performed by either wet chemical or plasmaprocesses. (See, for example, Angell et al. (37) and Manz et al. (38).

FIGS. 7A-7D illustrate the initial steps in microfabricating thediscrimination region portion of a nucleic acid sorting device (e.g.Device 20 in FIG. 1) by photolithographic techniques. As shown, thestructure includes a silicon substrate 160. The silicon wafer whichforms the substrate is typically washed in a 4:1H₂SO₄/H₂O bath, rinsedin water and spun dry. A layer 162 of silicon dioxide, preferably about0.5 μm in thickness, is formed on the silicon, typically by heating thesilicon wafer to 800-1200° C. in an atmosphere of steam. The oxide layeris then coated with a photoresist layer 164, preferably about 1 μminch-thickness. Suitable negative or positive resist materials are wellknown. Common negative resist materials include two-componentbisarylazide/rubber resists. Positive resist materials includepolymethyl-methacrylate (PMMA) and two component diazoquinone/phenol isresin materials. See, e.g., “introduction to microlithography”, Thompson(36).

The coated laminate is irradiated through a photomask 166 imprinted witha pattern corresponding in size and layout to the desired pattern of themicrochannels. Methods for forming photomasks having desired photomaskpatterns are well known. For example, the mask can be prepared byprinting the desired layout on an overhead transparency using a highresolution (3000 dpi) printer. Exposure is carried out on standardequipment such as a Karl Suss contact lithography machine.

In the method illustrated in FIGS. 7A-5D, the photoresist is a negativeresist, meaning that exposure of the resist to a selected wavelength,e.g., UV, light produces a chemical change that renders the exposedresist material resistant to the subsequent etching step. Treatment witha suitable etchant removes the unexposed areas of the resist, leaving apattern of bare and resist-coated silicon oxide on the wafer surface,corresponding to the layout and dimensions of the desiredmicrostructures. En this example, because a negative resist was used,the bare areas correspond to the printed layout on the photomask. Thewafer is now treated with a second etchant material, such as a reactiveion etch (RIE), effective to dissolve the exposed areas of silicondioxide. The remaining resist is removed, typically with hot aqueousH₂SO₄. The remaining pattern of silicon dioxide (162) now serves as amask for the silicon (160). The channels are etched in the unmaskedareas of the silicon substrate by treating with a KOH etching solution.Depth of etching is controlled by time of treatment. Additionalmicrocomponents may also be formed within the channels by furtherphotolithography and etching steps, as discussed below.

Depending on the method to be used for directing the flow of moleculesthrough the device, electrodes and/or valves are fabricated into theflow channels. A number of different techniques are available forapplying thin metal coatings to a substrate in a desired pattern. Theseare reviewed in, for example, Krutenat, Kirk-Othmer 3rd ed., Vol. 15,pp. 241-274 (32), incorporated herein by reference. A convenient andcommon technique used in fabrication of microelectronic circuitry isvacuum deposition. For example, metal electrodes or contacts may beevaporated onto a substrate using vacuum deposition and a contact maskmade from, e.g., a “MYLAR” sheet. Various metals such as platinum, gold,silver or indium/tin oxide (ITO) may be used for the electrodes.

Deposition techniques allowing precise control of the area of depositionare preferred for application of electrodes to the side walls of thechannels in the device. Such techniques are described, for example, inKrutenat (32), above, and references cited therein. They include plasmaspraying, where a plasma gun accelerates molten metal particles in acarrier gas towards the substrate, and physical vapor deposition usingan electron beam, where atoms are delivered on line-of-sight to thesubstrate from a virtual point source. In laser coating, a laser isfocused onto the target point on the substrate, and a carrier gasprojects powdered coating material into the beam, so that the moltenparticles are accelerated toward the substrate.

Another technique allowing precise targeting uses an electron beam toinduce selective decomposition of a previously deposited substance, suchas a metal salt, to a metal. This technique has been used to producesub-micron circuit paths (e.g., 26).

6.9. Elastomeric Microfabricated Device

This Example demonstrates the manufacture of a disposablemicrofabricated device, which can function as a stand-alone device or asa component of an integrated microanalytical chip, in sorting moleculesor cells.

Preparation of the microfabricated device. A silicon wafer was etchedand fabricated as described above and in (15). After standard contactphotolithography techniques to pattern the oxide surface of the siliconwafer, a C₂F₂/CHF₃ gas mixture was used to etch the wafer by RIE. Thesilicon wafer was then subjected to further etch with KOH to expose thesilicon underneath the oxide layer, thereby forming a mold for thesilicone elastomer. The silicon mold forms a “T” arrangement ofchannels. The dimensions of the channels may range broadly, havingapproximately 5×4 μm dimension.

The etching process is shown schematically in FIG. 8. Standardmicromachining techniques were used to create a negative master mold outof a silicon wafer. The disposable silicone elastomer chip was made bymixing General Electric RTV 615 components (20) together and pouringonto the etched silicon wafer. After curing in an oven for two hours at80° C., the elastomer was peeled from the wafer and bonded hermeticallyto a glass cover slip for sorting. To make the elastomer hydrophilic theelastomer chip was immersed in HCl (pH=2.7) at 60 degrees C. for 40 to60 min. Alternatively, the surface could have been coated withpolyurethane (3% w/v in 95% ethanol and diluted 10× in ethanol). It isnoted that the master wafer can be reused indefinitely. The device shownhas channels that are 100 μm wide at the wells, narrowing to 3 μm at thesorting junction (discrimination region). The channel depth is 4 μm, andthe wells are 2 mm in diameter. These dimensions can be modifiedaccording to the size range of the molecules or cells to be analyzed orsorted.

Detection Apparatus. In this embodiment the device was mounted on aninverted optical microscope (Zeiss Axiovert 35) as shown in FIG. 9. Inthis system, the flow control can be provided by voltage electrodes forelectro-osmotic control or by capillaries for pressure-driven control.The detection system can be photomultiplier tubes or photodiodes,depending upon the application. The inlet well and two collection wellswere incorporated into the elastomer chip on three sides of the “T”forming three channels (FIG. 7). The chip was adhered to a glasscoverslip and mounted onto the microscope.

6.10. Operation of a Polynucleotide Analysis Device

The operation of a polynucleotide analysis chip is described. Thisexample refers to polynucleotides, but it will be appreciated that othermolecules may be analyzed or sorted using similar methods and devices.Likewise, cells can be processed using similar methods and devices,adapted to the appropriate size.

A solution of reporter-labeled polynucleotides is prepared as describedbelow and introduced into the sample inlet end(s) of the analysisunit(s). The solution may be conveniently introduced into a reservoir,such as reservoir 48 of FIG. 1, via a port or connector, such asconnector 70 in FIG. 2, adapted for attachment to a segment of tubing,such as liquid chromatography or HPLC tubing.

It is typically advantageous to “hydrate” the device (i.e., fill thechannels of the device with the solvent, e.g., water or a buffersolution, in which the polynucleotides will be suspended) prior tointroducing the polynucleotide-containing solution. Such hydrating canbe achieved by supplying water or the buffer solution to the devicereservoir and applying hydrostatic pressure to force the fluid throughthe analysis unit(s).

Following such hydration, the polynucleotide-containing solution isintroduced into the sample inlets of the analysis unit(s) of the device.As the stream of labeled polynucleotides (e.g., tagged with a reportersuch as a fluorescent dye) is passed in a single file manner through thedetection region, the optical signal (e.g., fluorescence) from theoptically-detectable reporter moieties on each molecule are quantitatedby an optical detector and converted into a number used in calculatingthe approximate length of polynucleotide in the detection region.

Exemplary reporter moieties, described below in reference to samplepreparation, include fluorescent moieties which can be excited to emitlight of characteristic wavelengths by an excitation light source.Fluorescent moieties have an advantage in that each molecule can emit alarge number of photons (e.g., upward of 106) in response to excitingradiation. Suitable light sources include lasers, laser diodes,high-intensity lamps, e.g., mercury lamps, and the like. In embodimentswhere a lamp is used, the channels are preferably shielded from thelight in all regions except the detection region, to avoid bleaching ofthe label. In embodiments where a laser is used, the laser can be set toscan across a set of detection regions from different analysis units.Other optically-detectable reporter moieties include chemiluminescentmoieties, which can be used without an excitation light source.

Where laser diodes are used as a light source, the diodes may bemicrofabricated into the same chip that contains the analysis units.Alternatively, the laser diodes may be incorporated into a second chip(laser diode chip; LDC) that is placed adjacent to the chip such thatthe laser light from the diodes shines on the detection regions. Thephotodiodes in the LDC are preferably placed at a spacing thatcorresponds to the spacing of the detection regions in the chip.

The level of reporter signal is measured using an optical detector, suchas a photodiode (e.g., an avalanche photodiode), a fiber-optic lightguide leading, e.g., to a photomultiplier tube, a microscope with a highnumerical apperature (N.A.) objective and an intensified video camera,such as a SIT camera, or the like. The detector may be microfabricatedor placed into the PAC itself (e.g., a photodiode as illustrated, inFIGS. 3A and 3B), or it may be a separate element, such as a microscopeobjective.

In cases where the optical detector is a separate element, it isgenerally necessary to restrict the collection of signal from thedetection region of a single analysis unit. It may also be advantageousto scan or move the detector relative to the polynucleotide analysisunit (“PAC”); preferably by automation. For example, the PAC can besecured in a movable mount (e.g., a motorized/computer-controlledmicromanipulator) and scanned under the objective. A fluorescencemicroscope, which has the advantage of a built-in excitation lightsource (epifluorescence), is preferably employed for detection of afluorescent reporter.

Since current microfabrication technology enables the creation ofsub-micron structures employing the elements described herein, thedimensions of the detection region are influenced primarily by the sizeof the molecules under study. These molecules can be rather large bymolecular standards. For example, lambda DNA (˜50 kb) in solution has adiameter of approximately 0.5 μm. Accordingly, detection regions usedfor detecting polynucleotides in this size range have a cross-sectionalarea large enough to allow such a molecule to pass through without beingsubstantially slowed down relative to the flow of the solution carryingit and causing a “bottle neck”. The dimensions of a channel shouldtherefore be at least about twice, preferably at least about five timesas large per side or in diameter as the diameter of the largest moleculethat will be passing through it.

Another factor important to consider in the practice of the presentinvention is the optimal concentration of polynucleotides in the samplesolution. The concentration should be dilute enough so that a largemajority of the polynucleotide molecules pass through the detectionregion one by one, with only a small statistical chance that two or moremolecules pass through the region simultaneously. This is to insure thatfor the large majority of measurements, the level of reporter measuredin the detection region corresponds to a single molecule, rather thantwo individual molecules.

The parameters which govern this relationship are the volume of thedetection region and the concentration of molecules in the samplesolution. The probability that the detection region will contain two ormore molecules (P_(≧2)) can be expressed as

P _(≧2)=1−{1+[DNA]*V}*e ^(−{DNA}*V)

where [DNA] is the concentration of polynucleotides in units ofmolecules per μm³ and V is the volume of the detection region in unitsof μm³.

It will be appreciated that P_(≧2) can be minimized by decreasing theconcentration of polynucleotides in the sample solution. However,decreasing the concentration of polynucleotides in the sample solutionalso results in increased volume of solution processed through thedevice and can result in longer run times. Accordingly, the objectivesof minimizing the simultaneous presence of multiple molecules in thedetection chamber (to increase the accuracy of the sorting) needs to bebalanced with the objective of generating a sorted sample in areasonable time in a reasonable volume containing an acceptableconcentration of polynucleotide molecules.

The maximum tolerable P_(≧2) depends on the desired “purity” of thesorted sample. The “purity” in this case refers to the fraction ofsorted polynucleotides that are in the specified size range, and isinversely proportional to P_(≧2).

For example, in applications where high purity is not required, such asthe purification of a particular restriction fragment from an enzymaticdigest of a portion of vector DNA, a relatively high P_(≧2) (e.g.,P_(≧2)=0.2) may be acceptable. For most applications, maintaining P_(≧2)at or below about 0.1 provides satisfactory results.

In an example where P_(≧2) is equal 0.1, it is expected that in about10% of measurements, the signal from the detection region will be due tothe presence of two or more polynucleotide molecules. If the totalsignal from these molecules is in the range corresponding to the desiredsize fragment, these (smaller) molecules will be sorted into the channelor tube containing the desired size fragments.

The DNA concentration needed to achieve a particular value P_(≧2) in aparticular detection volume can be calculated from the above equation.For example, a detection region in the shape of a cube 1 μm³ per sidehas a volume of 1 femtoliter (fl). A concentration of moleculesresulting, on average, in one molecule per fl, is about 1.7 nM. Using aP_(≧2) of about 0.1, the polynucleotide concentration in a sampleanalyzed or processed using such a 1 fl detection region volume isapproximately 0.85 nM, or roughly one DNA molecule per 2 detectionvolumes ([DNA]*V=˜0.5). If the concentration of DNA is such that [DNA]*Vis 0.1, P_(≧2) is less than 0.005; i.e., there is less than a one halfof one percent chance that the detection region will at any given timecontain two of more fragments.

The signal from the optical detector is routed, e.g., via electricaltraces and pins on the chip, to a processor, which processes the signalsinto values corresponding to the length of the polynucleotide givingrise to the signal. These values are then compared, by the processor, topre-loaded instructions containing information on which branch channelmolecules of a particular size range will be routed into. Following adelay period that allows the molecule from which the reporter signaloriginated to arrive at the discrimination region, the processor sends asignal to actuate the active elements in the discrimination region suchthat the molecule is routed into the appropriate branch channel.

The delay period is determined by the rate at which the molecules movethrough the channel (their velocity relative to the walls of thechannel) and the length of the channel between the detection region andthe discrimination region. In cases where the sample solution is movedthrough the device using hydrostatic pressure (applied, e.g., aspressure at the inlet end and/or suction at the outlet end), thevelocity is typically the flow rate of the solution. In cases where themolecules are pulled through the device using some other means, such asvia electro-osmotic flow with an electric field set up between the inletend and the outlet end, the velocity as a function of molecule size canbe determined empirically by running standards, and the velocity for aspecific molecule calculated based on the size calculated for it fromthe reporter signal measurement.

A relevant consideration with respect to the velocity at which thepolynucleotide molecules move through the device is the shear force thatthey may be subject to. At the channel dimensions contemplated herein,the flow through the channels of the device is primarily laminar flowwith an approximately parabolic velocity profile. Since thecross-sectional area of the channels in the device can be on the sameorder of magnitude as the diameter of the molecules being analyzed,situations may arise where a portion of a particular molecule is verynear the wall of the channel, and is therefore in a low-velocity region,while another portion of the molecule is near the center of the channel,i.e., in a high-velocity region. This situation creates a shear force(F) on the molecule, which can be estimated using the followingexpression:

F=6πηR _(λ) V

where R_(λ) is the radius of the molecule and u is the viscosity of thesolution. This expression assumes that the molecule is immobilized on astationary surface and subject to uniform flow of velocity V.

The amount of force necessary to break a double stranded fragment of DNAis approximately 100 pN. Accordingly, the maximal shear force that themolecules are subjected to should preferably be kept below this value.Substituting appropriate values for the variables in the aboveexpression for lambda DNA yields a maximum velocity of about 1 cm/secfor a channel 1 μm in radius (i.e., a channel of a dimension where oneportion of the lambda molecule can be at or near the wall of the channelwith the opposite side in the center of the channel). Since devicesdesigned for use with such large molecules will typically have channelsthat are considerably larger in diameter, the maximum “safe” velocitywill typically be greater than 1 cm/sec.

As discussed above, the sample solution introduced into a device of theinvention should be dilute enough such that there is a high likelihoodthat only a single molecule occupies the detection region at any giventime. It follows then that as the solution flows through the devicebetween the detection and discrimination regions, the molecules will bein “single file” separated by stretches of polynucleotide-free solution.The length of the channel between the detection and discriminationregion should therefore not be so long as to allow random thermaldiffusion to substantially alter the spacing between the molecules. Inparticular, the length should be short enough that it can be traversedin a time short enough such that even the smallest molecules beinganalyzed will typically not be able to diffuse and “switch places” inthe line of molecules.

The diffusion constant of a 1 kb molecule is approximately 5 μm²/sec;the diffusion equation gives the distance that the molecule diffuses intime t as:

<X ² >˜Dt

Using this relationship, it can be appreciated that a 1 kbp fragmenttakes about 0.2 seconds to diffuse 1 μm. The average spacing ofmolecules in the channel is a function of the cross-sectional area ofthe channel and the molecule concentration, the latter being typicallydetermined in view of acceptable values of P_(≧2) (see above). From theabove relationships, it is then straightforward to calculate the maximumchannel length between the detection and discrimination region whichwould ensure that molecules don't “switch places”. In practice, thechannel length between the detection and discrimination regions isbetween about 1 μm and about 2 cm.

As illustrated above with respect to FIGS. 5A-D, there are a number ofways in which molecules can be routed or sorted into a selected branchchannel. For example, in a device employing the discrimination regionshown in FIG. 4A, the solution is preferably moved through the device byhydrostatic pressure. Absent any field applied across electrodes 110 and112, a molecule would have an equal probability of entering one or theother of the two branch channels 106 and 108. The sorting isaccomplished by the processor temporarily activating a voltage sourceconnected to the electrode leads 114 and 116 just before or at the timethe molecule to be routed enters the junction of the main channel andthe two branch channels. The resulting electric field exerts a force onthe negatively-charged DNA molecule biasing it toward thepositively-charged electrode. The molecule will then be carried down thebranch channel containing the positively-charged electrode by the bulksolution flow. The electric field is turned off when the molecule hascommitted itself to the selected channel. As soon as the molecule clearsthe corner from the discrimination region and into the branch channel,it escapes effects of the electric field that will be applied to thenext molecule in the solution stream.

The discrimination region shown in FIG. 5B is designed for use in adevice that employs electroosmotic flow, rather than flow induced byhydrostatic pressure, to move both the polynucleotides and bulk solutionthrough the device. Electrodes are setup in the channels at the inletand outlet ends of the device. Application of an electric field at theends of the channels (with electrode 130 being negative, and electrodes132 and 134 being positive) sets up bulk solution flow according towell-established principles of electroosmotic flow (see, e.g., 25). Whena specific polynucleotide molecule enters the junction region betweenthe main channel and the two branch channels, the voltage to one ofeither electrodes 132 or 134 is shut off, leaving a single attractiveforce, acting on the solution and the DNA molecule, into the selectedbranch channel. As above, both branch channel electrodes are activatedafter the molecule has committed to the selected branch channel in orderto continue bulk flow through both channels.

In another embodiment of the invention the polynucleotides are directedinto a selected branch channel via a valve in the discrimination region.An exemplary valve is shown in FIG. 5C. The valve consists of a thinextension of material 140 which can be charged via an electrode 142. Theextension can then be deflected to close one or the other of the branchchannels by application of an appropriate voltage across electrodes 144and 146. As above, once the molecule has committed, the voltage can beturned off.

In a device in which the sample solution is moved through the device byapplication of positive pressure at the sample inlet end(s) of theanalysis unit(s), the discrimination function may be affected by simplyblocking branch channel sample outlets into which the sample is notsupposed to go, and leaving the selected outlet open. Due to the smallsize scale of the channels and the incompressibility of liquids,blocking the solution flow creates an effective “plug” in the unselectedbranch channels, routing the molecule along with the bulk solution flowinto the selected channel. This embodiment is illustrated in FIG. 4D. Itcan be achieved by, for example, incorporating valve structuresdownstream of the discrimination region.

Alternatively, the discrimination function may be affected by changingthe hydrostatic pressure at the sample outlets of the branch channelsinto which the sample is not supposed to go. Specifically, if the branchchannels in a particular analysis unit all offer the same resistance tofluid flow, and the pressure at the sample inlet of the main-channel ofan analysis unit is P, then the fluid flow out of any selected branchchannel can be stopped by applying a pressure P/n at the sample outletof that branch channel, where n is the number of branch channels in thatanalysis unit. Accordingly, in an analysis unit having two branchchannels, the pressure applied at the outlet of the branch to be blockedis P/2.

It will be appreciated that the position and fate of the molecules inthe discrimination region can be monitored by additional detectionregions installed, e.g., immediately upstream of the discriminationregion and/or in the branch channels immediately downstream of thebranch point. This information be used by the processor to continuouslyrevise estimates of the velocity of the molecules in the channels and toconfirm that molecules having selected size characteristics end up inthe selected branch channel.

Solution from the branch channels is collected at the outlet ends of theanalysis units. As described above, devices with a plurality of analysisunits typically collect the solution from corresponding branch channelsof each unit into a manifold, which routes the solution flow to anoutlet port, which can be adapted for receiving, e.g., a segment oftubing or a sample tube, such as a standard 1.5 ml centrifuge tube.

The time required to isolate a desired quantity of polynucleotidedepends on a number of factors, including the size of thepolynucleotide, the rate at which each analysis unit can process theindividual fragments, and the number of analysis units per chip, and canbe easily calculated using basic formulas. For example, a chipcontaining 1000 analysis units, each of which can sort 1000 fragmentsper second, could isolate 0.1 μg of 10 kb DNA in about 2.5 hours.

6.11. Other Microfabricated Devices

Operation of a microfabricated cell sorting device is essentially asdescribed above with respect to the polynucleotide sorting device. Sincecells typically do not have predictable a net charge, the directingmeans are preferably ones employing a valve in the discrimination regionas described above, or flow stoppage, either by valve or hydrostaticpressure.

Operation of a microfabricated analysis device is accomplishedessentially as is described above, except that functions relating tosorting polynucleotide molecules into branch channels don't need to beperformed. The processor of such analysis devices is typically connectedto a data storage unit, such as computer memory, hard disk or the like,as well as to a data output unit, such as a display monitor, printerand/or plotter. The sizes of the polynucleotide molecules passingthrough the detection region are calculated and stored in the datastorage unit. This information can then be further processed and/orrouted to the data output unit for presentation as, e.g., histograms ofthe size distribution of DNA molecules in the sample. The data can, ofcourse, be presented in real time as the sample is flowing through thedevice, allowing the practitioner of the invention to continue the runonly as long as is necessary to obtain the desired information.

In preferred molecular (e.g. DNA, polynucleotide or polypeptide)analysis and sorting embodiments, a microfabricated chip of theinvention has a detection volume of about 10 to about 5000 femtoliters(fl), preferably about 50 to about 1000 fl, and most preferably on theorder of about 200 fl. In preferred cell analysis and sortingembodiments, a microfabricated chip of the invention has a detectionvolume of approximately 1 to 1,000,000 femtoliters (fl), preferablyabout 200 to 500 fl, and most preferably about 375 fl.

6.12. Exemplary Embodiment and Additional Parameters

Microfluidic Chip Fabrication. In a preferred embodiment, the inventionprovides a “T” on “Y” shaped series of channels molded into opticallytransparent silicone rubber or PolyDiMethylSitoxane (PDMS), preferablyPDMS. This is cast from a mold made by etching the negative image ofthese channels into the same type of crystalline silicon wafer used insemiconductor fabrication. As described above, the same techniques forpatterning semiconductor features are used to form the pattern of thechannels. The uncured liquid silicone rubber is poured onto these moldsplaced in the bottom of a Petri dish. To speed the curing, these pouredmolds are baked. After the PDMS has cured, it is removed from on top ofthe mold and trimmed. In a chip with one set of channels forming a “T”,three holes are cut into the silicone rubber at the ends of the “T”, forexample using a hole cutter similar to that used for cutting holes incork, and sometimes called cork borers. These holes form the sample,waste and collection wells in the completed device. In this example, thehole at the bottom end of the T is used to load the sample. The hole atone arm of the T is used for collecting the sorted sample while theopposite arm is treated as waste. Before use, the PDMS device is placedin a hot bath of HCl to make the surface hydrophilic. The device is thenplaced onto a No. 1 (150 μm thick) (25×25 mm) square microscope coverslip. The cover slip forms the floor (or the roof) for all threechannels and wells. The chip has a detection region as described above.

Any of or all of these manufacturing and preparation steps can be doneby hand, or they can be automated, as can the operation and use of thedevice.

The above assembly is placed on an inverted Zeiss microscope. A carrierholds the cover slip so that it can be manipulated by the microscope'sx-y positioning mechanism. This carrier also has mounting surfaces whichsupport three electrodes, which implement the electro-osmotic and/orelectrophoretic manipulation of the cells or particles to be analyzedand sorted. The electrodes are lengths of platinum wire taped onto asmall piece of glass cut from a microscope slide. The wire is bent intoa hook shape, which allows it to reach into one of the wells from above.The cut glass acts as a support platform for each of the electrodes.They are attached to the custom carrier with double-sided tape. Thisallows flexible positioning of the electrodes. Platinum wire ispreferred for its low rate of consumption (long life) in electrophoreticand electro-osmotic applications, although other metals such as goldwire may also be used.

Device Loading. To operate the device for sorting, one of the wells,e.g. the collection or waste well, is first filled with buffer. Allthree channels, starting with the channel connected to the filled well,wick in buffer via capillary action and gravity. Preferably, no otherwell is loaded until all the channels fill with buffer, to avoid theformation of air pockets. After the channels fill the remaining wellscan be loaded with buffer, as needed, to fill or equilibrate the device.The input or sample well is typically loaded last so that the flow ofliquid in the channels is initially directed towards it. Generally,equal volumes of buffer or sample are loaded into each well. This isdone in order to prevent a net flow of liquid in any direction once allof the wells are loaded, including loading the sample well with sample.In this embodiment, it is preferred that the flow of material throughthe device (i.e. the flow of sample) be driven only by the electrodes,e.g. using electro-osmotic and/or electrophoretic forces. The electrodesmay be in place during loading, or they can be placed into the wellsafter loading, to contact the buffer.

Electrodes. Two of the above electrodes are driven by high voltageoperational amplifiers (op-amps) capable of supplying voltages of +−150V. The third electrode is connected to the electrical ground (or zerovolts) of the high voltage op-amp electronics. For sorting operation thedriven electrodes are placed in the collection and waste wells. Theground electrode is placed in the sample well. The op-amps amplify, by afactor of 30, a control voltage generated by two digital to analogconverters (DACs). The maximum voltage these DACs generate is +−5 V,which determines the amplification factor of 30. The 150 V limit isdetermined by the power supply to the amplifiers, which are rated for+−175 V. These DACs reside on a card (a Lab PC 1200 Card, available fromNational Instruments, Austin, Tex.) mounted in a personal computer. Thecard also contains multiple channels of analog to digital converters(ADC's) one of which is used for measuring the signal generated byphotomultiplier tubes (PMTs). This card contains two DACs. A third DACcan be used to drive the third electrode with an additional high voltageop amp. This would provide a larger voltage gradient, if desired, andsome additional operational flexibility.

Without being bound by any theory, it is believed that the electrodesdrive the flow of sample through the device using electro-osmotic orelectrophoretic forces, or both. To start the movement of molecules,cells or particles to be sorted, a voltage gradient is established inthe channels. This is done by generating a voltage difference betweenelectrodes.

In this example, the voltage of the two driven electrodes is raised orlowered with respect to the grounded electrode. The voltage polaritydepends on the charge of the molecules, cells or particles to be sorted(if they are charged), on the ions in the buffer, and on the desireddirection of flow. Because the electrode at the sample well in theexamples is always at zero volts with respect to the other twoelectrodes, the voltage at the “T” intersection or branch point will beat a voltage above or below zero volts, whenever the voltage of theother two electrodes is raised or lowered. Typically, the voltage is setor optimized, usually empirically, to produce a flow from the samplewell, toward a downstream junction or branch point where two or morechannels meet. In this example, where two channels are used, one channelis typically a waste channel and terminates in a waste well; the otherchannel is a collection channel and terminates in a collection well.

To direct the molecules, particles or cells to a particular channel orarm of the “T” (e.g. collection or waste), the voltage at the electrodein one well (or multiple wells, in multi-channel embodiments) is madethe same as the voltage at the junction or branch point, where thechannels meet. The voltage of the electrode at one well of the two ormore wells is raised or lowered, to produce a gradient between that welland the branch point. This causes the flow to move down the channeltowards and into the well, in the direction produced by the gradient.Typically, the voltage of the electrode at the waste well is raised orlowered with respect to the voltage at the collecting well, to directthe flow into the waste channel and the waste well, until a molecule,particle or cell is identified for collection. The flow is diverted intothe collection channel and collection well by adjusting the voltages atthe electrodes to eliminate or reduce the gradient toward the wastewell, and provide or increase the gradient toward the collection well.For example, in response to a signal indicating that a molecule or cellhas been detected for sorting, by examination in a detection regionupstream of the branch point, the voltage at the waste and collectionpoints can be switched, to divert the flow from one channel and well tothe other.

The voltage at the branch point (the intersection voltage) is determinedby the voltage gradient desired (e.g. Volts/mm) times the distance fromthe sample well electrode to the branch point (gradient×distance), whichin this example is placed where all of the channels of the “T”intersect. The gradient also determines the voltage at the waste orcollection electrode(gradient×distance from sample well to collectionwell).

Conceptually, the channels and wells of the “T” can be treated as anetwork of three resistors. Each segment of the “T” behaves as aresistor whose resistance is determined by the conductivity of thebuffer and the dimensions of the channel. A voltage difference isprovided across two of the resistors, but not the third. If theelectrodes in each of the three wells is equidistant from the branchpoint, then each channel will have the same resistance.

For example, assume that each section of the “T” has 100 K ohms ofresistance. If 100 volts is applied across two of the resistors and thethird resistor is left unconnected, the current at the junction of thetwo resistors would be 50 volts. If a voltage source of 50 volts isconnected to the end of the third resistor, the voltage at the junctiondoes not change. That is, a net of zero volts is established across thethird resistor; there is no voltage gradient and a flow is not initiatedor changed. If a different voltage is applied, a gradient can beestablished to initiate or direct the flow into one channel or another.For example, to change the direction of flow from one arm of the “T” tothe other, the voltage values of the two driven electrodes are swapped.The junction voltage remains the same. If the electrode distances fromthe “T” intersection are not equal, then the voltages can be adjusted toaccommodate the resulting differences in the effective channelresistance. The end result is still the same. The electrode in the wellof the channel which is temporarily designated not to receive particlesor cells is set at the voltage of the“T” intersection. The voltage atthe other driven electrode is set to provide a gradient that directsmolecule, cell or particle flow into that well. Thus, cells or particlescan be sent down one channel or another; and ultimately into one well oranother, by effectively opening one channel with a net or relativevoltage gradient while keeping the other channel or channels closed by anet or relative voltage gradient of zero.

In a preferred embodiment for sorting according to the invention, aslight flow down the channel that is turned “off” is desired. This keepsthe molecules or cells moving away from the branch point (the “T”junction), particularly those which have already been directed to thatchannel. Thus, a small non-zero gradient is preferably established inthe “off” or unselected channel. The selected channel is provided with asignificantly higher gradient, to quickly and effectively divert thedesired molecules or cells into that channel.

The placement of the wells and their electrodes with respect to thebranch point, and in particular their distance from the branch point, isan important factor in driving the flow of molecules or cells asdesired. As the wells and electrodes are brought closer to the branchpoint, it becomes more important to precisely place the electrodes, orprecisely adjust the voltages.

Detection Optics. En this example, a Ziess Axiovert 35 invertedmicroscope is used for detection of molecules or cells for sorting. Theobjective lens of this microscope faces up, and is directed at thedetection region of the described microfluidic chip, through thecoverslip which in this example is the floor of the device. Thismicroscope contains all the components for epifluorescence microscopy.See, Inoue pp 67-70, 91-97 (52). In this embodiment a mercury arc lampor argon ion laser is used as the light source. The mercury lampprovides a broad spectrum of light that can excite many differentfluorophores. The argon ion laser has greater intensity, which improvesthe detection sensitivity but is generally restricted to fluorophoresthat excite at 488 or 514 nm. The mercury tamp is used, for example, tosort beads as described elsewhere herein. The laser is used for sortingGFP E. coli bacterial cells as described elsewhere herein. The highpower argon ion beam is expanded to fill the illumination port of themicroscope, which matches the optical characteristics of the mercury arclamp and provides a fairly uniform illumination of the entire image areain a manner similar to the mercury lamp. However, it is somewhatwasteful of the laser light. If a lower powered laser is used, the laserlight is focused down to coincide with the detection region of the chip,to achieve the same or similar illumination intensity and uniformitywith less power consumption.

The objective used in the example is an Olympus PlanApo 60×1.4 N.A. oilimmersion lens. The optics are of the infinity corrected type. An oilimmersion lens enables collecting a substantial percentage of the 180degree hemisphere of emitted light from the sample. This enables some ofthe highest sensitivity possible in fluorescence detection. Thismicroscope has 4 optical ports including the ocular view port. Eachport, except the ocular, taps ˜20% of the available light collected fromthe sample when switched into the optical path. Only the ocular port canview 100% of the light collected by the objective. In this embodiment, acolor video camera is mounted on one port, another has a Zeissadjustable slit whose total light output is measured with aphotomultiplier tube (PMT). The fourth port is not used.

The microscope focuses the image of the sample onto the plane of theadjustable slit. An achromatic lens collimates the light from the slitimage onto the active area of the PMT. Immediately in front of the PMTwindow an optical band pass filter is placed specific to thefluorescence to be detected. The PMT is a side on-type and does not havea highly uniform sensitivity across its active area. By relaying theimage to the PMT with the achromat, this non-uniformity is averaged andits effect on the measured signal is greatly minimized. This alsoenables near ideal performance of the bandpass filter. A 20% beamsplitter has been placed in the light path between the achromat andfilter. An ocular with a reticle re-images this portion of thecollimated light. This enables viewing the adjustable slit directly, toinsure that the detection area that the PMT measures is in focus andaligned. The adjustable slit allows windowing a specific area of thechannel for detection. Its width, height, and x,y position areadjustable, and conceptually define a detection region on the chip. Inthis embodiment, the microscope is typically set to view a 5 μm (micron)length of the channel directly below the “T” intersection.

The PMT is a current output device. The current is proportional to theamount of light incident on the photocathode. A transimpedance amplifierconverts this photo-current to a voltage that is digitized by the Lab PC1200 card. This allows for interpreting the image to select cells orparticles having an optical reporter for sorting, as they pass throughthe detection region, based for example on the amount of light orfluorescence measured as an indication of whether a cell or particle hasa predetermined level of reporter and should be chosen for collection.Voltages at the electrodes of the chip can be adjusted or switchedaccording to this determination, for example by signals initiated by orunder the control of a personal computer acting in concert with the LabPC1200 card.

Absorbence Detection. In another embodiment for detecting cells ormolecules, absorbence detection is employed, which typically usesrelatively longer wavelengths of light, e.g., ultraviolet (UV). Thus,for example, a UV light source can be employed. Additional objectivelenses can be used to image a detection region, such that the lenses arepreferably positioned from the top surface if the PDMS device is madereasonably thin. Measurement of the light transmitted, i.e., notabsorbed by the particle or cell, using an adjustable slit, e.g., aZeiss adjustable slit as described above, is similar to that used influorescence detection. A spectrophotometer may also be used. Asmolecules, particles or cells pass through the detection window theyattenuate the light, permitting detection of a desired characteristic orthe lack thereof. This can improve the accuracy of the particle sorting,for example, when sorting based on an amount of a characteristic, ratherthan presence of the characteristic alone, or to confirm a signal.

It is noted that in some cases, detection by absorbence may bedetrimental at certain wavelengths of light to some biological material,e.g., E. coli cells at shorter (UV) wavelengths. Therefore, biologicalmaterial to be sorted in this manner should first be tested first undervarious wavelengths of light using routine methods in the art.Preferably, a longer wavelength can be selected which does not damagethe biological material of interest, but is sufficiently absorbed fordetection.

Optical trapping. In another embodiment, an optical trap, or lasertweezers, may be used to sort or direct molecules or cells in a PDMSdevice of the invention. One exemplary method to accomplish this is toprepare an optical trap, methods for which are well known in the art,that is focused at the “T” intersection proximate to, and preferablydownstream of, the detection region. Different pressure gradients areestablished in each branch. A larger gradient at one branch channelcreates a dominant flow of molecules, particles or cells, which shouldbe directed into the waste channel. A second, smaller gradient atanother branch channel should be established to create a slower flow offluid from the “T” intersection to another channel for collection. Theoptical trap remains in an “off” mode until a desired particle isdetected at the detection region. After detection of a desiredcharacteristic, the particle or cell is “trapped”, and thereby directedor moved into the predetermined branch channel for collection. Themolecule or cell is released after it is committed to the collectionchannel by turning off the trap laser. The movement of the cell ormolecule is further controlled by the flow into the collection well. Theoptical trap retains its focus on the “T” intersection until thedetection region detects the next molecule, cell or particle.

Flow control by optical trapping permits similar flexibility in bufferselection as a pressure driven system. In addition, the pressuregradients can be easily established by adjusting the volume of liquidadded to the wells. However, it is noted that the flow rate will not beas fast when the pressure in one channel is above ambient pressure andpressure in another is below.

Forward Sorting. In an electrode-driven embodiment, prior to loading thewells with sample and buffer and placing the electrodes, the electrodevoltages are set to zero. Once the sample is loaded and the electrodesplaced, voltages for the driven electrodes are set, for example usingcomputer control with software that prompts for the desired voltages,for example the voltage differential between the sample and wasteelectrodes. If the three wells are equidistant from the “T”intersection, one voltage will be slightly more than half the other. Ina typical run, the voltages are set by the program to start withdirecting the molecules, particles or cells to the waste channel. Theuser is prompted for the threshold voltage of the PMT signal, toidentify a molecule, particle or cell for sorting, i.e. diversion to thecollection channel and well. A delay time is also set. If the PMTvoltage exceeds the set threshold, the driven electrode voltages areswapped and then, after the specified delay time, the voltages areswapped back. The delay allows the selected molecule, particle or cellenough time to travel down the collection channel so that it will not beredirected or lost when the voltages are switched back. As describedabove, a slight gradient is maintained in the waste channel; when thevoltages are switched, to provide continuity in the flow. This is notstrong enough to keep the molecule, particle or cell moving into theother or “off” channel it if is too close to or is still at the branchpoint.

The value of this delay depends primarily on the velocity of themolecules, particles or cells, which is usually linearly dependent onthe voltage gradients. It is believed that momentum effects do notinfluence the delay time or the sorting process. The molecules,particles or cells change direction almost instantaneously with changesin the direction of the voltage gradients. Unexpectedly, experimentshave so far failed to vary the voltages faster than the particles orcells can respond. Similarly, experiments have so far shown that thedimensions of the channels do not effect the delay, on the distance andtime scales described, and using the described electronics. In additionthe speed with which the cells change direction even at high voltagegradients is significantly less than needed to move them down theappropriate channel, when using a forward sorting algorithm.

Once the voltage and delay value are entered the program, it enters asorting loop, in which the ADC of the Lab PC 1200 card is polled untilthe threshold value is exceeded. During that time, the flow of particlesor cells is directed into one of the channels, typically a wastechannel. Once the threshold is detected, the above voltage switchingsequence is initiated. This directs a selected cell or particle (usuallyand most preferably one at a time) into the other channel, typically acollection channel. It will be appreciated that the cells or particlesare being sorted and separated according to the threshold criteria,without regard for which channel or well is considered “waste” or“collection”. Thus, molecules or cells can be removed from a sample forfurther use, or they can be discarded as impurities in the sample.

After the switching cycle is complete (i.e. after the delay), theprogram returns to the ADC polling loop. A counter has also beenimplemented in the switching sequence which keeps track of the number oftimes the switching sequence is executed during one run of the program.This should represent the number of molecules, cells or particlesdetected and sorted. However, there is a statistical chance that twomolecules, cells or particles can pass through simultaneously and becounted as one. In this embodiment, the program continues in thispolling loop indefinitely until the user exits the loop, e.g. by typinga key on the computer keyboard. This sets the DACs (and the electrodes)to zero volts, and the sorting process stops.

Reverse Sorting. The reverse sorting program is similar to the forwardsorting program, and provides additional flexibility and an errorcorrection resource. In the event of a significant delay in changing thedirection of flow in response to a signal to divert a selectedmolecules, cell or particle, for example due to momentum effects,reversible sorting can change the overall direction of flow to recoverand redirect a molecule, cell or particle that is initially divertedinto the wrong channel. Experiments using the described electrode arrayshow a delay problem and an error rate that are low enough (i.e.virtually non-existent), so that reversible sorting does not appearnecessary. The algorithm and method may be beneficial, however, forother embodiments such as those using pressure driven flow, which thoughbenefitting from an avoidance of high voltages, may be more susceptibleto momentum effects.

If a molecule or cell is detected for separation from the flow, andswitching is not fast enough, the molecule or cell will end up goingdown the waste channel with all of the other undistinguished cells.However, if the flow is stopped as soon as possible after detection, themolecule or cell will not go too far. A lower driving force can then beused to slowly drive the particle in the reverse direction back into thedetection window. Once detected for a second time, the flow can bechanged again, this time directing the molecule or cell to thecollection channel. Having captured the desired molecule or cell, thehigher speed flow can be resumed until the next cell is detected forsorting. This is achieved by altering the voltages at the electrodes, oraltering the analogous pressure gradient, according to the principlesdescribed above.

To move molecules or cells at higher velocities, for faster and moreefficient sorting, higher voltages may be needed, which could bedamaging to molecules or cells, and can be fatal to living cells.Preliminary experiments indicate that there may be a limit to thetrade-off of voltage and speed in an electrode driven system.Consequently, a pressure driven flow may be advantageous for certainembodiments and applications of the invention. Reversible sorting may beadvantageous or preferred in a pressure driven system, as hydraulic flowswitching may not be done as rapidly as voltage switching. However, if amain or waste flow can move fast enough, there may be a net gain inspeed or efficiency over voltage switching even though the flow istemporarily reversed and slowed to provide accurate sorting. Pressuredriven applications may also offer wider flexibility in the use ofbuffers or carriers for sample flow, for example because a response toelectrodes is not needed.

6.13. Diagnosis of Tuberculosis

A method for DNA fingerprinting is disclosed, and is particularlysuitable for forensic identification (e.g. by VNTR), bacterial typingand humam or animal pathogen diagnosis. The method applies restrictionlength polymorphism using synthetically generated polynucleotidefragments. This method may be used with PCR, but in preferredembodiments PCR is not required.

In this example, the invention is applied to diagnosing the presence ofthe tuberculosis bacteria (TB). A short sequence of the TB genome, e.g.20-50 bp (base pairs), is selected that is a fixed distance from arestriction site. This sequence and its relationship to the restrictionsite are known or statistically predicted to be unique or stronglycharacteristic of TB and can serve to distinguish TB from otherorganisms, alone or in combination with other sequences and/orrestriction sites. Thus, additional short sequences can be selected inrelation to the same or different restriction sites, in order toincrease statistical discrimination. In this way, a unique fingerprintof DNA fragments can be constructed.

To identify the bacteria, a single-stranded DNA oligolnucleotide issynthesized to complement the sequence of each short fragment. Thesample DNA to be identified is denatured, and is combined with theoligonucleotides, triphosphates and DNA polymerase. Some of thenucleotides should be fluorescently labeled to serve as a reporter. Astrand of fluorescent DNA will be synthesized, which can be cut at therestriction site to yield a fragment of fixed length. The resulting DNAfragments can then be sized in any suitable way, for example by getelectrophoresis, or the microfabricated technologies described herein,or both. The use of micro fabricated devices is preferred. These devicesare fast (10 minutes) and require only femtograms of sample DNA, i.e.only a few thousand molecules. If multiple oligonucleotides are made,the reactions can be multiplexed, for example all of theoligonucleotides can be combined with the sample DNA in one test tube.

6.14. DNA Fingerprinting

This example demonstrates construction a synthetic fingerprint of a DNAsample and identification of a sample. The method can be varied in anumber of ways that will be apparent to the practitioner. For example,post-staining with an intercalating dye can be used in place of labelednucleotides. Suitable dyes include those that are specific to doublestranded polynucleotides or DNA (e.g. picogreen from Molecular Probes,Inc.). Alternatively, single stranded DNA can be digested, for exampleusing a single-strand specific nuclease. Another variation is to useaffinity purification to pull down the fragment of interest, for exampleusing biotinylated oligonucleotides and streptavidin magnetic coatedbeads.

DNA samples are denatured or digested with a specific restrictionenzyme, such as Bgl II, EcoR I, Hind III or Xho I in the presence of abuffer, according to the instructions from the manufacturer. This can bedone at about 34° C. for about 1 minute. Multiple digestions may be doneand a final mixture of thousands of base pairs is preferred. The DNAfragments are then extended with primers and fluorescent nucleotides. Ifthe buffer used for digestion is not compatible with DNA extension,another buffer may be used, or dialysis or ethanol precipitation can beconducted.

The DNA extension is accomplished by first preparing a 10× primersolution (1 μM) and a 10× nucleotide mix comprising 250 μM dATP, 500 μMfluorescein-dATP (from NEN), and 750 μM dTTP, dCTP and dGTP. The DNAsample is then diluted in TE buffer to get a final 1.0× concentration ofabout 5-50 ng/μl. This strongly depends on the average size of DNAfragments in the digestion sample and also the relative amount of DNAfragments (templates) that will hybridize with the primers. Generallyspeaking, about 1 nM of DNA templates (10×) gives optimum results. DNAextension can be done at about 68° C. for about 2 hours.

The following solutions are made:

Mix 2 (Gibco Elongase Enzyme Mix) Mix 1 (total 100 μl) 2 μl ultra-purewater 16 μl 5X Buffer A 1 μl of 10x nucleotide mix 24 μl Buffer B 1 μlof 10x primer  4 μl Elongase Enzyme Mix (Gibco) 1 μl of 10x DNA samples56 μl of ultra-pure waterBuffer A and Buffer B are from the Gibco Elongase Enzyme Package. Then,combine equal volumes of Mix 1 and Mix 2 and mix well. 100 μl of Mix 2can be used for about 20 different DNA samples (in Mix 1). Therefore,the total volume of Mix 2 can be adjusted according to the number of DNAsamples to be run.

Following these procedures, a polymerase reaction is run for 1 minute at96° C. for denaturing, for 1 minute at 55° C. for annealing and for 1hour at 68° C. for extension. The annealing time and temperature mayvary, depending on the primer used in Mix 1. The PCR reaction andconditions can be optimized or modified according to techniques known inthe art.

Preferably, only one reaction is performed. Successive rounds of PCRamplification are not needed. Dialysis or a spin column is then used toclean out unused fluorescent single nucleotides (fluorescein-dATP).

After the extension reaction is run the fluorescent-labeled DNA samplesare diluted to about 100 fM (1,000 times for a 10× template with aconcentraion of 1 nM). Then 10 μl of the diluted sample is run on theSMS system described above, using 10 mW laser power and 2,450-volt APDbias for 10 to 30 minutes. The data is analyzed using a DNA sizedistribution or threshold method according to a pre-selected fluorescentlevel, as described above.

6.15. Identification of T7 DNA

This example demonstrates construction of a synthetic fingerprint fromthe genome of T7 phage, and identification of a sample, according to themethods of Example 14. An oligonuceotide primer specific to T7 phage wasused, fluorescent fragments were generated, and these were sized in anSMS device of the invention. Control tests using the T7 olignucleotideprimer as the fingerprint with a lambda phage sample showed virtually nosignal in the SMS device of Example 9.

En this example, with T7 and λ, digestion was not needed and was notdone, because the DNA fragments were already of fixed length. The DNAfragments are extended with primers and fluorescent nucleotides.Dialysis was done at 4° C. for 3 hours using a 5 μl sample in 10 ml ofTE buffer. After dialysis a 10× dilution in TE buffer was done (i.e. byadding 45 μl of TE buffer).

The DNA extension is accomplished by first preparing a 10× primersolution 1 μM and a 10× nucleotide mix comprising 250 μM dATP, 500 μMfluorescein-dATP (from NEN), and 750 μM dTTP, dCTP and dGTP. The DNAsample is then diluted in TE buffer to get a final 10× concentration ofabout 5-50 ng/μl. This strongly depends on the average size of DNAfragments in the digestion sample and also the relative amount of DNAfragments (templates) that will hybridize with the primers. Generallyspeaking, about 1 nM of DNA templates (10×) gives optimum results. DNAextension can be done at about 68° C. for about 2 hours.

Mix 1 and Mix 2 were made as in Example 14. Then, 5 μl of Mix 2 wasadded to Mix 1 and they are mixed well.

In this example the following T7 primer was used, which binds to T7 atbase position 588:

(SEQ. ID. NO. 1) 5′-CATTGACAACATGAAGTAACATGCAGTAAGA-3′.

Following these procedures, a polymerase reaction is run for 1 minute at96° C. for denaturing, for 1 minute at 60° C. for annealing and for 2hours at 68° C. for extension. In this example, 0.5 μl of a 100 μlsolution of Taq polymerase was used. Only one reaction is performed.Successive rounds of PCR amplification are not needed. Dialysis or spincolumn is then used to clean out unused fluorescent single nucleotides(fluorescein-dATP). After the extension reaction is run thefluorescent-labeled DNA samples are diluted to about 100 fM. Then 10 μlof the diluted sample is run on the SMS system, using 10 mW laser powerand 2,450-volt APD bias for 10 to 30 minutes. The data is analyzed usingDNA size distribution or threshold method according to a pre-selectedfluorescent level.

For analysis using a microfabricated device of the invention, a samplesolution was diluted by a factor of two. The device comprised a T-shapedset of channels about 3 μm wide and about 1.75 μm deep in an elastomersubstrate. This chip was treated with LICE to render it hydrophillic.The device, as described in Example 9, used a laser with 3.5 mW of power(488 nm) with a 2,450 V APD detector bias.

The above procedure was repeated with an unknown test sample, whichcomprised either T7 phage DNA (which should match) or lambda phage(which should not match). The sizing results for the test sample werecompared against the T7 phage standard. The results of the T7 v. T7experiment are shown in FIG. 13. The results of the T7 v. lambda phageexperiment are shown in FIG. 14. These results are compared in FIG. 15,showing strong signals for the T7 matching T7, and weak signals forlambda, which does not match T7. As shown, there is a clearly adistinguishable signal between the T7 and lambda results. FIG. 13 showsmany brightly fluorescent DNA fragments that are relatively long(match), compared to the relatively short and dull fragments of FIG. 14(no match). These differences can be readily detected based on a presetthreshold, as shown in FIG. 15. The results shown in the figures wereobtained with 5-10 minutes of run time on the SMS device of theinvention.

6.16. Preferred Systems and Embodiments for Molecular Fingerprinting

This Example describes, in general terms, preferred embodiments of themolecular fingerprinting assays that are described and demonstratedelsewhere in this application (see, for instance, the Examples presentedin Sections 6.13-6.15, supra). The description of these methods is madeby way of non-limiting examples. Accordingly, the skilled artisan willappreciate that many variations of these methods may be practicedwithout departing from the spirit of the present invention. For example,many of the specific steps described above may be eliminated and,moreover, the steps need not necessarily be performed in the particularsequential order(s) recited herein.

Using the methods described herein, a skilled artisan may readilyanalyze any sample, e.g., to detect a particular nucleic acid and tothereby determine whether that particular nucleic acid is present in thesample. For example, the methods of the invention may be used to analyzesamples of cells or tissue (or samples of nucleic acid derivedtherefrom) to determine whether a particular nucleic acid is present inthe cells or tissue. In one embodiment, the methods may be used todetermine whether the cells or tissue express a particular nucleic acid.In another embodiment the particular nucleic acid may be a nucleic acidfrom a pathogen (e.g., from an infectious agent such as a virus orbacteria), and the fingerprinting methods of this invention may be usedto determine whether the cells or tissues are infected with thatpathogen.

In other embodiments, the methods of the invention may be used toanalyze a sample derived from an individual (e.g., a clinical samplederived from a patient). For example, the molecular fingerprintingmethods of this invention may be used to analyze a cell or tissue samplefrom an individual or, more preferably, a sample or nucleic acidsderived from such cells or tissue. In such embodiments the molecularfingerprinting methods of the invention may be used, e.g., as adiagnostic method (e.g., to detect a nucleic acid or nucleic acidscharacteristic of a particular pathogen), as part of a therapeuticmethods (e.g., to monitor expression of a certain gene or genes during atherapy) or as a forensic method (for example, paternity testing) toname a few applications.

In preferred embodiments, the methods of this invention are used todetect or analyze single stranded nucleic acids within a sample.Accordingly, a nucleic acid sample analyzed according to the inventionwill preferably be a sample of single-stranded nucleic acid molecules.However, samples containing double-stranded nucleic acids may also beanalyzed. In such embodiments, the sample is preferably denatures (e.g.,by heat) or otherwise exposed to conditions in which the complementarynucleic acids separate to produce single-stranded nucleic acid.

The skilled artisan will readily appreciate that the methods of thisinvention may be used to analyze and/or detect any type of nucleic acidin a sample. Thus, although the specific examples presented hereinfrequently describe the invention in terms of detecting or analyzingDNA, the invention may also be used to detect and/or analyze RNA.Indeed, any type of synthetic or naturally occurring nucleic acid may beanalyzed and/or detected using the invention, including but not limitedto the various types recited, supra, in Section 5.1.

In preferred embodiments, therefore, the invention is used to detect aparticular nucleic acid, referred to here as the “target” nucleic acid,in a sample. Preferably, the base sequence of the target nucleic aciddetected with this invention will be at least partly known. Morepreferably, the known (or partly known) sequence will comprise at leastone sequence that is recognized by a particular “cleavage agent”. Theterm cleavage agent, as used herein, refers to any agent (e.g., anyenzyme, chemical or other substance) that is able to cut or cleave anucleic acid molecule into two or more fragments. In preferredembodiments, each cleavage agent used in the methods and compositions ofthis invention will recognize a specific (and preferably different)nucleic acid sequence; i.e., each cleavage agent preferably has aspecific recognition site (also referred to herein as the “cleavagesite” or the “restriction site”). Recognition sites that are four basesin length are preferred. However, the invention is not limited torecognition sites of any particular length. Indeed, in embodiments wherea plurality of different cleavage agents are used, the differentcleavage agents may have recognition sites of different lengths.

In particularly preferred embodiments, a cleavage agent used in theinvention is a restriction enzyme. Restriction enzymes are well known inthe art and may be readily obtained, e.g., from a variety of commercialsources (for example, Promega Corp., Madison, Wis.). Similarly, methodsfor using restriction enzymes are also generally well known and routinein the art. See, for example, the references cited in Section 5.1,supra, for general molecular biology techniques. Preferred restrictionenzymes are ones that cut nucleic acids by recognizing a specificsequence of bases (i.e., the recognition site). Typically, therecognition site for a restriction enzyme will be about 4, 5 or 6nucleotides in length. A cut is typically made within the recognitionsite. Therefore the location of the cut in a nucleic acid having a knownrecognition site will also be known.

In one preferred, exemplary embodiment of the invention, a primer isselected or chosen (e.g., by a user) which is able to bind or hybridizeto the target nucleic acid under suitable conditions and at a specific,known or predetermined location in the target nucleic acid sequence. Inparticular, the primer preferably binds or hybridizes to the targetnucleic acid at a location that is a known or predetermined distancefrom the restriction site; i.e., at a site that is a specific number ofbases away from the restriction site. Generally, the primer is a nucleicacid that is complementary to a particular sequence of the targetnucleic acid molecule and is therefore capable of hybridizing to thatcomplementary sequence under appropriate hybridization conditions. Inpreferred embodiments, the primer is an oligonucleotides between about 4and about 100 bases in length, more typically about 10-100 bases inlength and preferably between about 20-50 bases in length.

A suitable primer having been selected, chosen or otherwise obtained,the primer is then contacted to the nucleic acid sample under suitableconditions so that the primer binds to the target nucleic acid at thepredetermined location. The sample, with the primer, is then incubatedwith a polymerase and a plurality of nucleotides under conditions suchthat primer extension can occur, e.g., by adding nucleotides to theprimer and using the target nucleic acid as a template, therebygenerating a second nucleic acid molecule that is complementary to thetarget nucleic acid. Typically, the plurality of nucleotides willinclude one or more nucleotides that are detectably labeled (e.g., witha reporter) so that the primer extension product, or a fragment thereof,may be detected by detecting the reporter.

In one embodiment, the primer extension reaction continues to the end ofthe target nucleic acid. However, in preferred embodiments primerextension need only continue up to at least the restriction site so thatthe complementary nucleic acid that is generated by the primer extensionalso contains the restriction site. Generally, polymerases synthesizepolynucleotides beginning at the 3′-end of a polynucleotide primer andmoving in the 3′-direction. Thus, in preferred embodiments of theinvention, a primer is chosen that hybridizes to a sequence of thetarget nucleic acid that is situated a particular known distanceupstream (i.e., in the 3′-direction) from the restriction site on thesingle-stranded target nucleic acid.

Having generated a nucleic acid that is complementary to the targetnucleic acid and contains the target nucleic acid's restriction site (ora complement thereof), the complementary nucleic acid may also be cut orcleaved by a cleavage agent (e.g., a restriction enzyme) whichrecognizes the particular restriction site. Because synthesis of thecomplementary nucleic acid beings a fixed, predetermined distance fromthe restriction site, using the cleavage agent to cut or cleave thecomplementary nucleic acid gives rise to a fragment having a knownlength; namely, the length of the known, fixed distance from therestriction site to the position on the target sequence where the primerhybridizes and primer extension begins.

Accordingly, in preferred embodiments the cleavage agent is nextcontacted to the sample so that the target sequence and/or thecomplementary sequence are cut or cleaved, thereby generating a fragmentor fragments having the fixed known length. In one embodiment, thecleavage agent is contact to the sample without denaturing thepolynucleotides; i.e., so that the target and complementary nucleicacids are hybridized to each other and are therefore double stranded.However, in an alternative embodiment the cleavage agent is able tospecifically recognize the restriction site in the single-strandedcomplementary nucleic acid, and the two strands are separated (e.g., byheating the sample) before contacting the cleavage agent.

A user may then readily determine whether the target nucleic acid ispresent in the sample by detecting fragments of the known fixed length.For instance, in preferred embodiments where primer extension isperformed using detectably labeled nucleotides (e.g., with a reporter),the fragments may be readily detected by separating polynucleotides inthe sample according to length and detecting the reporter. A variety oftechniques are known in the art for separating polynucleotides by theirlength or size and any of these techniques may be used in the presentinvention. Exemplary, non-limiting techniques include gelelectrophoresis, high performance liquid chromatography (HPLC) and massspectroscopy. However, in particularly preferred embodimentspolynucleotides are sorted according to size using a microfluidic deviceof the present invention, e.g., according to any of the polynucleotidesorting algorithms described supra.

A skilled artisan will recognize that the above-described methods may bereadily adapted to various nucleic acid amplification techniques such asthe polymerase chain reaction (PCR). For example, many different copiesof a single primer may be repeatedly contacted to the sample followed byrepeated primer extension reactions so that a plurality of complementarynucleic acids are obtained. Alternatively, sense and antisense primersmay be designed to amplify a particular subsequence of the targetnucleic acid which contains the restriction site. Contacting thecleavage agent with the resulting amplification products will thenproduce fragments having a specific, predetermined size and thesefragments may be detected as described above.

The methods of the invention are ideally suited however, to performingonly a single round of primer extension so that a single complementarynucleic acid is created and a single fragment is detected. Singlenucleic acid fragments may be readily detected, e.g., using amicrofluidic device of the invention to sort polynucleotides onemolecular at a time, as described supra. Moreover, these methods areparticularly advantageous since analysis may be performed using smallsamples and without undergoing complicated thermocycling that isrequired, e.g., for PCR. As result, the molecular fingerprinting methodsof this invention are ideally suited for “lab on a chip” devices(described supra) that comprise a single microfluidic device. In suchdevices, the entire sequence of primer extension, strand cleavage andfragment detection may be performed using a single microfluidic deviceor kit and such kits are therefore considered to be part of the presentinvention.

A skilled artisan will also readily appreciate that steps of thesemolecular fingerprinting methods may be performed in a variety ofdifferent sequences. For example, in one alternative embodiment thenucleic acid sample may be contacted with a cleavage agent beforeimplementing primer extension. In such embodiments, the primer will thenhybridize to a nucleic acid fragment generated when the target nucleicacid is cut or cleaved with the cleavage agent. In particular, theprimer preferably hybridizes to this fragment a known, predetermineddistance from the fragment's end (i.e., from the cut site). As a result,primer extension occurs from the primer and along the target sequence tothe cut site, and a complementary fragment having the known,predetermined length is thereby obtained.

Those skilled in the art will further appreciate that variations of theabove-described methods may be performed using a plurality of differentprimers. Preferably, each primer will hybridize to a different sequencewithin the target nucleic acid, each different sequence being a knownbut different distance from the target nucleic acids restriction site.In such variations of the invention, a plurality of primer extensionreactions may be implemented (preferably at least one for each differentprimer) either sequentially or at the same time, and thedifferent-extension products may then be cleaved with the cleavageagent. A plurality of fragments having different predetermined lengthsare then produced, and these fragments may be separated according totheir size and detected as described, above.

It will be appreciated by persons of ordinary skill in the art that theexamples and preferred embodiments herein are illustrative, and that theinvention may be practiced in a variety of embodiments which share thesame inventive concept.

7. REFERENCE CITED

Numerous references, including patents, patent applications and variouspublications, are cited and discussed in the description of thisinvention. The citation and/or discussion of such references is providedmerely to clarify the description of the present invention and is not anadmission that any such reference is “prior art” to the inventiondescribed herein. ALL references cited and discussed in thisspecification and/or listed here below are incorporated herein byreference in their entirety and to the same extent as if each referencewas individually incorporated by reference.

BIBLIOGRAPHY

-   1. J. P. Nolan, L. A. Sklar, Nature Biotechnology 16,633 (1998).-   2. P. J. Crosland-Taylor, Nature (London) 171, 37 (1953).-   3. U.S. Pat. No. 2,656,508 issued to Coulter (1949).-   4. L. A. Kamensky, M. R. Melamed, H. Derman, Science 150, 630    (1965).-   5. A. Moldavan, Science 80, 188 (1934).-   6. M. A. Van Villa, T. T. Trujillo, P. F. Mullaney, Science 163,    1213 (1969).-   7. M. A. Van Villa, et al., A fluorescent cell photometer: a new    method for the rapid measurement of biological cells stained with    fluorescent dyes. (Biological and Medical Research Group of the    Health Division, LASL., 1997).-   8. M. J. Fulwyer, Science 156, 910 (1974).-   9. H. M. Shapiro, Practical Flow Cytometry (Wiley-Liss Inc., New    York City, 1995).-   10. M. R. Melamed, T. Lindmo, M. L. Mendelsohn, Flow Cytometry and    Sorting (Wiley-Liss Inc., New York City, 1990).-   11. G. Whitesides, Y. Xia, Angewandte Chemie International Edition    37, 550 (1998).-   12. P. H. Li, D. J. Harrison, Analytical, Chemistry 69, 1564 (1997).-   13. S. Fiedler, et al. Analytical Chemistry 70, 1909-1915 (1998).-   14. L. A. Sklar, Proc. SPIE 3256, 144 (1998).-   15. H. P. Chou, A. Scherer, C. Spence, S. R. Quake, Proc. Natl.    Acad. Sci. USA 96: 11-13 (1998).-   16. A. Ashkin, J. M. Dziedzic, Science 235, 1517 (1987).-   17. A. Ashkin, J. M. Dziedzic, Nature 330, 769 (1987).-   18. T. N. Buican, M. J. Smyth, H. A. Verissman, Applied Optics 26,    5311 (1987).-   19. C. Spence, S. R. Quake, “Transformation of cells with DNA    sorting on microchips.”; personal communication, 1998.-   20. R. V. Hare, “Polyvinylsiloxane impression material.”; U.S. Pat.    No. 5,661,222, 1997.-   21. M. U. Kopp et al., Science, 280: 1046 (1998)-   22. D. J. Harrison et al., Science, 261: 895 (1993)-   23. J. P. Brody, “Valveless Microswitch, U.S. Pat. No. 5,656,155    (1998).-   24. Aine, H. E., et al., U.S. Pat. No. 4,585,209 (1986).-   25. Baker, D. R., in Capillary Electrophoresis, John Wiley Sons, New    York, 1995.-   26. Ballantyne, J. P., et al., J. Vac. Sci. Technol. 10:1094 (1973).-   27. Castro, A., et al., Anal. Chem. 85:849-852 (1993).-   28. Goodwin, P. M., et: al., Nucleic Acids Research 21(4):803-806    (1993).-   29. Gravesen, P., et: al., U.S. Pat. No. 5,452,878 (1995).-   30. Haugland, R. P., in Handbook of Fluorescent Probes and Research    Chemicals, 5th Ed., Molecular Probes, Inc., Eugene, Oreg. (1992).-   31. Keller, R. A., et al., GB Patent No. 2,264,296 (10/95).-   32. Krutenat, R. C., Kirk-Othmer Concise Encyclopedia of Chemical    Technology, John Wiley & Sons, New York (1985).-   33. O'Connor, J. M., U.S. Pat. No. 4,581,624 (1986).-   34. van Lintel, H. T. G., U.S. Pat. No. 5,271,274 (1993).-   35. Wise, K. D., et al., U.S. Pat. No. 5,417,235 (1995).-   36. Thompson, L. F., “Introduction to Lithography”, ACS Symposium    Series 219:1-13, (1983).-   37. Angell et al., Scientific American 248:44-55 (1983).-   38. Manz et al.; Trends in Analytical Chemistry 10: 144-149 (1991)-   39. Harrison et al., International Publication No. 98/52691,    published Nov. 26, 1998.-   40. Bein, Thomas, Efficient Assays for Combinatorial Methods for the    Discovery of Catalysts, Angew. Chem. Int. Ed. 38:3, 323-26 (1999).-   41. F. H. Arnold, Acct. Chem. Research 31, 125-131 (1998).-   42. Hanes, J. & Pluckthun A. Proc. Natl. Acad. Sci., USA 94, 4937    (1997).-   43. Hoffmuller, U. & J. Schneider-Mergener, Angew. Chemie. Int. Ed.    37, 3241-3243 (1998).-   44. Jermutus, L., L. A. Ryabova & A. Pluckthun, Curr. Opin.    Biotechnol. 9, 534-548 (1998).-   45. Roberts, R. W. & Szostak, J. W. Proc. Natl. Acad. Sci. USA 94,    12297-12302 (1997).-   46. Stemmer, W. P. C. Nature, 370, 389 (1994).-   47. Tawfik, D. and Griffiths, A. Nat. Biotechnol. 16, 656 (1998).-   48. Sambrook et at., Molecular Cloning: A Laboratory Manual 2^(nd)    Edition, Cold Spring Harbor Laboratory Press (1989).-   49. Benecke et al., U.S. Pat. No. 5,454,472 (1995).-   50. J. Affholter and F. Arnold, “Engineering a Revolution,”    Chemistry in Britain, April 1999, p. 48.-   51. H. Joo, Z. Lin and F. Arnold, “Laboratory evolution of    peroxide-mediated cytochrome P450 hydroxylation,” Nature 399,    670-673 (1999).-   52. Inoue, Shinya and Spring, Kenneth R., Video Microscopy: The    Fundamentals, 2nd ed., Plenum Press, New York, N.Y. (1997).-   53. Giusti, J. Forensic Sci. 31:409-417 (1986).-   54. Kanter et al, J. Forensic Sci. 31:403-408 (1986).-   55. Jeffreys et al., Nature 314:67-72 (1985).-   56. Budowle et al., Am. J. Hum. Genet. 48:137-144 (1991).-   57. Nakamura et al., Science 235:1616-22 (1987).-   58. Crow et al., “The Evaluation of Forensic DNA Evidence,” National    Academy Press (1996).-   59. Quake et al., “A Microfabricated Device for Sizing and Sorting    DNA Molecules,” PNAS 96:11-13 (1999).

1. A molecular fingerprinting method comprising the steps of: (a)identifying a target polynucleotide, (b) selecting at least one fragmentof the target polynucleotide, wherein the fragment is a fixed distancefrom a restriction site, to generate a set of one or more polynucleotidefragments, and (c) designating some or all of the set of fragments asmolecular fingerprint corresponding to the target polynucleotide. 2-55.(canceled)